O-Mannosyltransferase Deficient Filamentous Fungal Cells and Methods of Use Thereof

ABSTRACT

The present disclosure relates to compositions and methods useful for the production of heterologous proteins with reduced O-mannosylation in filamentous fungal cells, such as Trichoderma cells. More specifically, the invention provides a PMT-deficient filamentous fungal cell comprising a) at least a first mutation that reduces an endogenous protease activity compared to a parental filamentous fungal cell which does not have said first mutation, and, b) at least a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation, wherein said filamentous fungal cell is selected from the group consisting of Trichoderma, Neurospora, Myceliophthora or Chrysosporium cell.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods useful forthe production of heterologous proteins in filamentous fungal cells.

BACKGROUND

Posttranslational modification of eukaryotic proteins, particularlytherapeutic proteins such as immunoglobulins, is often necessary forproper protein folding and function. Because standard prokaryoticexpression systems lack the proper machinery necessary for suchmodifications, alternative expression systems have to be used inproduction of these therapeutic proteins. Even where eukaryotic proteinsdo not have posttranslational modifications, prokaryotic expressionsystems often lack necessary chaperone proteins required for properfolding. Yeast and fungi are attractive options for expressing proteinsas they can be easily grown at a large scale in simple media, whichallows low production costs, and yeast and fungi have posttranslationalmachinery and chaperones that perform similar functions as found inmammalian cells. Moreover, tools are available to manipulate therelatively simple genetic makeup of yeast and fungal cells as well asmore complex eukaryotic cells such as mammalian or insect cells (DePourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31).

However, posttranslational modifications occurring in yeast and fungimay still be a concern for the production of recombinant therapeuticprotein. In particular, O-mannosylation is one of the biggest hurdles toovercome in the production of biopharmaceuticals for human applicationsin fungi. More specifically, yeasts like Pichia pastoris andSaccharomyces cerevisiae tend to hyper-mannosylate heterologouslyexpressed biopharmaceuticals, thereby triggering adverse effects whenapplied to humans.

O-mannosylation to Serine and Threonine residues includes in mammalsGalNAc based oligosaccharides or GlcNAc/N-acetyllactosamine comprisingO-linked mannose glycans. In fungi O-mannosylation occurs as hexosemonomers or oligomers. In yeasts, there are typically severalprotein(/polypeptide) O-mannosyltransferases, which often function ascomplexes. Part of the knock-outs are harmfull, at least for cellstructures and stability and not all yeast knock-outs or combinationsare tolerated (for a review, see Goto 2007, Biosci. Biotechnol. Biochem.71(6), 1415-1427).

There have been reports of knock-outs of yeast O-mannosyltransferasegenes, aiming to reduce the O-mannosylation levels, and even multipleknock-out mutants involving two or three pmt genes in S. cerevisiae(WO/1994/004687). Pmt1 or pmt2 knock-out of S. cerevisiae reduced thelevel of O-mannosylation of antifreeze glycoprotein III to about 30% ofthe proteins and the residual mannosylated protein contains numerousmannose residues per protein, apparently also oligosaccharides(WO/2004/057007).

WO/2010/034708 reports no significant level of O-mannosylation ofrecombinant hydrophobin Trichoderma protein when expressed in pmt1knock-out of S. cerevisiae host cell. Such O-mannosylation appears to beartificial yeast glycosylation of the original non-mannosylatedfilamentous fungal protein.

WO/2010/128143 further reports single chain antibody-albumin fusionconstruct in yeast S. cerevisiae pmt1 and/or pmt4 knock-out strains.

Pmt1, pmt2, and pmt3 single gene knock-outs, double, and tripleknock-outs of Aspergillus species (Aspergillus nidulans, Aspergillusfumigatus, and/or Aspergillus awamori) are described in Goto et al, 2009(Eukaryotic cell 2009, 8(10):1465); Mouyna et al, 2010 (MolecularMicrobiology 2010, 76(5), 1205-1221); Zhou et al, 2007 (Eukaryotic cell2007, 6(12):2260); Oka et al, 2004 (Microbiology 2004, 150, 1973-1982);Kriangkripipat et al, 2009; Fang et al, 2010 (Glycobiology, 2010, vol.20 pp 542-552); and Oka et al, 2005 (Microbiology 2005, 151, 3657-3667).

Despite numerous reports on knock out of pmt homologues in filamentousfungi, there is no description of a filamentous fungal cell with reducedO-mannosylation and useful as a host cell for the production ofrecombinant glycoprotein.

In particular, Gorka-Niec et al (2008, Acta Biochimica Polonica, Vol. 55No 2/2008, 251-259) reported the deletion of pmt1 gene in Trichodermareesei. PMT1 protein showed the highest identity to Pmt4p of S.cerevisiae (51%) but functionally complement pmt2Δ S. cerevisiae mutant(Gorka-Niec et al, 2007, Biochimica et Biophysica Acta 1770, 2007,774-780). However, the authors reported that disruption of the pmt1 genecaused a decrease of protein secretion but did not alter O- andN-glycosylation of secreted protein. Zakrzewska et al (Curr Genet 200343: 11-16) further reported that Trichoderma reesei pmt1 gene did notfunctionally complement pmt4Δ S. cerevisiae mutant.

In fact, deletions of the PMT genes in yeasts or filamentous fungiappears to either result in no phenotype at all or lethality or severelyimpaired vital functions of the cells, which would not be suitable forrecombinant production of heterologous proteins, especially mammalianglycoproteins. For this reason, alternative methods such as the use ofpmt inhibitors have been proposed as an alternative to pmt knock outstrains (WO2009/143041).

Thus, a need remains for improved filamentous fungal cells, such asTrichoderma fungus cells, that can stably produce heterologous proteinswith no or reduced O-mannosylation, such as immunoglobulins, preferablyat high levels of expression.

SUMMARY

The present invention relates to improved methods for producing proteinswith no or reduced O-mannosylation in filamentous fungal expressionsystems, and more specifically, glycoproteins, such as antibodies orrelated immunoglobulins or fusion proteins which may be O-mannosylatedwhen produced in filamentous fungal expression systems.

The present invention is based in part on the surprising discovery thatfilamentous fungal cells, such as Trichoderma cells, can be geneticallymodified to reduce or suppress O-mannosylation activity, withoutadversely affecting viability and yield of produced glycoproteins.

Accordingly, in a first aspect, the invention relates to a PMT-deficientfilamentous fungal cell comprising

-   a) a first mutation that reduces or eliminates an endogenous    protease activity compared to a parental filamentous fungal cell    which does not have said first mutation, and,-   b) a second mutation in a PMT gene that reduces endogenous    O-mannosyltransferase activity compared to a parental filamentous    fungal cell which does not have said second mutation, wherein said    filamentous fungal cell is selected from the group consisting of    Trichoderma, Neurospora, Myceliophthora and Chrysosporium cell.

In one embodiment, said PMT-deficient cell further expresses aheterologous protein containing serine and/or threonine residues. Theexpressed heterologous protein with serine and/or threonine residues hasreduced O-mannosylation due to said mutation in said PMT gene. Forexample, the O-mannosylation level of the heterologous protein expressedin a PMT-deficient cell of the invention is at least 10%, 20%, 30%, 40%,50%, 60%, 70%, 80% or at least 90% lower as compared to theO-mannosylation level of the heterologous protein when expressed in theparental filamentous fungal cell which does not have said secondPMT-deficient mutation.

In another embodiment, said second mutation that reduces endogenousO-mannosyltransferase activity is a deletion or a disruption of a PMTgene encoding an endogenous protein O-mannosyltransferase activity.

In another embodiment, said second PMT-deficient mutation in a PMT genemay be a mutation (such as a deletion or disruption) in either:

-   a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,-   b) a functional homologous gene of PMT1 gene, which functional gene    is capable of restoring parental O-mannosylation level by functional    complementation when introduced into a T. reesei strain having a    disruption in said PMT1 gene, or,-   c) a polynucleotide encoding a polypeptide having at least 50%, at    least 60%, at least 70%, at least 90%, or at least 95% identity with    SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.

In another embodiment that may be combined with the precedentembodiments, said PMT-deficient cell has a third mutation that reducesor eliminates the level of expression of an ALG3 gene compared to thelevel of expression in a parental cell which does not have such thirdmutation. In a specific embodiment, said PMT-deficient cell furthercomprises a first polynucleotide encodingN-acetylglucosaminyltransferase I catalytic domain and a secondpolynucleotide encoding N-acetylglucosaminyltransferase catalyticdomain.

In another embodiment that may be combined with the precedingembodiments, said PMT-deficient cell further comprises one or morepolynucleotides encoding a polypeptide selected from the groupconsisting of:

-   a) α1,2 mannosidase,-   b) N-acetylglucosaminyltransferase I catalytic domain,-   c) α mannosidase II, and-   d) N-acetylglucosaminyltransferase catalytic domain.

In another embodiment that may be combined with the precedingembodiments, said PMT-deficient cell further comprises one or morepolynucleotides encoding a β1,4 galactosyltransferase and/or afucosyltransferase.

In one specific embodiment, said PMT-deficient cell is a Trichodermacell comprising at least a mutation that reduces or eliminates theprotein O-mannosyltransferase activity of Trichoderma pmt1, and,optionally, further comprising mutations in at least one or more otherPMT genes that reduces or eliminates the protein O-mannosyltransferaseactivity selected from the group consisting of pmt2 and pmt3.

In one embodiment that may be combined with the preceding embodiments,the PMT deficient cells comprise mutations that reduce or eliminate theactivity of at least two, or at least three endogenous proteases.Typically, said cell may be a Trichoderma cell and may comprisemutations that reduce or eliminate the activity of

-   a) the three endogenous proteases pep1, tsp1 and slp1,-   b) the three endogenous proteases gap1, slp1 and pep1,-   c) three endogenous proteases selected from the group consisting of    pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2,    slp3, slp7, gap1 and gap2,-   d) three to six proteases selected from the group consisting of    pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2,    or,-   e) seven to ten proteases selected from the group consisting of    pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3,    slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.

In one embodiment that may be combined with the precedent embodiments,the filamentous fungal cell of the invention does not comprise adeletion or disruption of an endogenous gene encoding a chaperoneprotein. In particular, said filamentous fungal cell of the inventionexpresses functional endogenous chaperone protein Protein DisulphideIsomerase (PDI).

In another aspect, the invention relates to a method for producing aprotein having reduced O-mannosylation, comprising:

-   a) providing a PMT-deficient filamentous fungal cell, having a    mutation in a PMT gene that reduces endogenous O-mannosyltransferase    activity as compared to parental strain which does not have such    mutation, and further comprising a polynucleotide encoding a protein    with serine or threonine residue,-   b) culturing said PMT-deficient filamentous fungal cell to produce    said protein with reduced O-mannosylation,    wherein said filamentous fungal cell is selected from the group    consisting of Trichoderma, Neurospora, Myceliophthora and    Chrysosporium cell.

According to one specific embodiment of the method, said mutation in aPMT gene is a mutation, such as a deletion or disruption, in either:

-   a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,-   b) a functional homologous gene of PMT1 gene, which gene is capable    of restoring parental O-mannosylation level by functional    complementation when introduced into a T. reesei strain having a    disruption in said PMT1 gene, or,-   c) a polynucleotide encoding a polypeptide having at least 50%, at    least 60%, at least 70%, at least 90%, or at least 95% identity with    SEQ ID NO:2, said polypeptide having protein O-mannosyltransferase    activity.

In another embodiment of the method, said PMT-deficient cell is aTrichoderma reesei cell and said mutation is a deletion or a disruptionof T. reesei PMT1 gene.

In other embodiments of the method, said PMT-deficient cell is aPMT-deficient cell of the invention as described above.

In a specific embodiment, said polynucleotide encoding a protein is arecombinant polynucleotide encoding a heterologous protein. Typically,said heterologous protein may be a mammalian protein selected from thegroup consisting of

-   -   a) an immunoglubulin, such as IgG,    -   b) a light chain or heavy chain of an immunoglobulin,    -   c) a heavy chain or alight chain of an antibody,    -   d) a single chain antibody,    -   e) a camelid antibody,    -   f) a monomeric or multimeric single domain antibody,    -   g) a FAb-fragment, a FAb2-fragment, and,    -   h) their antigen-binding fragments.

In one embodiment of the method, that may be combined with the precedingembodiments, said polynucleotide encoding said protein further comprisesa polynucleotide encoding CBH1 catalytic domain and linker as a carrierprotein and/or cbh1 promoter.

In another embodiment, said polynucleotide encodes a protein with serineor threonine, which may be O-mannosylated in a PMT functional parentalstrain, and further comprising at least one N-glycan.

The invention also relates to a method for producing an antibody havingreduced O-mannosylation, comprising:

-   -   a) providing a PMT-deficient filamentous fungal cell having        -   i. a mutation that reduces endogenous protein            O-mannosyltransferase activity as compared to parental            strain which does not have such mutation and        -   ii. a polynucleotide encoding a light chain antibody and a            polynucleotide encoding a heavy chain antibody,    -   b) culturing the cell to produce said antibody, consisting of        heavy and light chains, having reduced O-mannosylation,    -   wherein said filamentous fungal cell is selected from the group        consisting of Trichoderma, Neurospora, Myceliophthora and        Chrysosporium cell.

In a specific method for producing antibody, said PMT-deficient cell isa Trichoderma reesei cell and said mutation is a deletion or adisruption of T. reesei PMT1 gene.

In one embodiment of the method for producing antibody, at least 70%,80%, 90%, 95%, or 100% of the produced antibody is not O-mannosylated.

The invention also relates to the protein composition or antibodycomposition obtainable or obtained by the methods of the invention asdescribed above. In one embodiment, at least 70%, 80%, 90%, 95%, or 100%of the antibodies as obtained or obtainable the methods of the inventionare not O-mannosylated.

In one specific embodiment, such protein (e.g. a glycoprotein) orantibody composition with reduced O-mannosylation comprises, as a majorglycoform, either,

-   -   Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (Man5 glycoform);    -   Manα6(Manα3)Manβ4GlcNAβ4GlcNAc (Man3 glycoform);    -   hybrid or complex type N-glycans such as glycoforms selected        from the subgroup consisting of GlcNAcMan3, G0, hybrid glycan,        or GlcNAcMan5, or galactosylated derivatives, such as        GalGlcNAcMan3, G1, G2; or, GalGlcNAcMan5 glycoform.

In one specific embodiment, when the core of the glycan consists ofMan3, then the composition essentially lacks Man5 glycoforms.

In an embodiment that may be combined with one or more of the precedingembodiments less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/orO-glycans of the protein composition comprises Neu5Gc and/or Galα−structure. In an embodiment that may be combined with the precedingembodiments, less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/orO-glycans of the antibody composition comprises Neu5Gc and/or Galα−structure.

In an embodiment that may be combined with one or more of the precedingembodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of theglycoprotein composition comprises core fucose structures. In anembodiment that may be combined with the preceding embodiments, lessthan 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of the antibodycomposition comprises core fucose structures.

In an embodiment that may be combined with one or more of the precedingembodiments, less than 0.1%, 0.01%, 0.001%, or 0% of N-glycan of theglycoprotein composition comprises terminal galactose epitopesGalβ3/4GlcNAc. In an embodiment that may be combined with the precedingembodiments, less than 0.1%, 0.01%, 0.001%, or 0% of the N-glycan of theantibody composition comprises terminal galactose epitopesGalβ3/4GlcNAc.

In an embodiment that may be combined with one or more of the precedingembodiments, less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of theglycoprotein composition comprises glycation structures. In anembodiment that may be combined with the preceding embodiments, lessthan 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the antibody compositioncomprises glycation structures.

In another embodiment that may be combined with one or more of thepreceding embodiments, the glycoprotein composition, such as an antibodyis devoid of one, two, three, four, five, or six of the structuresselected from the group of Neu5Gc, terminal Galα3Galβ4GlcNAc, terminalGalβ4GlcNAc, terminal Galβ3GlcNAc, core linked fucose and glycationstructures.

The invention also relates to a method of reducing O-mannosylation levelof a recombinant glycoprotein composition produced in a filamentousfungal cell, for example, Trichoderma cell, typically, Trichodermareesei, said method consisting of using a filamentous fungal cell havinga mutation in a PMT gene wherein said PMT gene is either:

-   -   i. PMT1 gene comprising the polynucleotide of SEQ ID NO:1,    -   ii. a functional homologous gene of PMT1 gene, which gene is        capable of restoring parental O-mannosylation level by        functional complementation when introduced into a T. reesei        strain having a disruption in said PMT1 gene, or,    -   iii. a polynucleotide encoding a polypeptide having at least        50%, at least 60%, at least 70%, at least 90%, or at least 95%        identity with SEQ ID NO:2, said polypeptide having protein        O-mannosyltransferase activity.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts results for Southern analyses of Trichoderma reesei pmt1deletion strains expressing antibody MAB01. A) A 5.7 kb signal isexpected from parental strains M124 and M304 with pmt1 ORF probe afterSpeI+XbaI digestion. No signal is expected from pure pmt1 deletionstrains. B) A 3.5 kb signal is expected for pmt1 5′flank probe fromdeletion strains after SpeI+AscI digestion. C) A 1.7 kb signal isexpected for pmt1 3′flank probe from deletion strains after AscI+XbaIdigestions. AscI does not cut intact pmt1 locus in close distance,therefore signals of over 16 kb (B) and 10 kb (C) are expected fromparental strains M124 or M304. A 4.1 kb signal is expected from PmeIdigested plasmid pTTv185 used as a control in hybridisations with bothflank probes (B, C).

FIG. 2 depicts a spectra of light chain of flask cultured parental T.reesei strain M317 (pyr4⁻ of M304) (A) and Δpmt1 disruptant clone 26-8A(B), day 7.

FIG. 3 depicts results for Western analyses of Trichoderma reesei pmt1deletion strain M403 from fed-batch fermentation. Upper panel: MAB01light chain, lower panel: MAB01 heavy chain. 0.1 μl of supernatant wasloaded on each lane.

FIG. 4 depicts a spectrum of light chain of fermenter cultured T. reeseistrain M403 (pmt1 deletion strain of MAB01 antibody producing strain,clone 26-8A), day 7.

FIG. 5 depicts a phylogeny of PMTs of selected filamentous fungi.

FIG. 6 depicts a partial sequence alignment of the results of the PMTBLAST searches.

DETAILED DESCRIPTION Definitions

As used herein, an “expression system” or a “host cell” refers to thecell that is genetically modified to enable the transcription,translation and proper folding of a polypeptide or a protein ofinterest, typically of mammalian protein.

The term “polynucleotide” or “oligonucleotide” or “nucleic acid” as usedherein typically refers to a polymer of at least two nucleotides joinedtogether by a phosphodiester bond and may consist of eitherribonucleotides or deoxynucleotides or their derivatives that can beintroduced into a host cell for genetic modification of such host cell.For example, a polynucleotide may encode a coding sequence of a protein,and/or comprise control or regulatory sequences of a coding sequence ofa protein, such as enhancer or promoter sequences or terminator. Apolynucleotide may for example comprise native coding sequence of a geneor their fragments, or variant sequences that have been optimized foroptimal gene expression in a specific host cell (for example to takeinto account codon bias).

As used herein, the term, “optimized” with reference to a polynucleotidemeans that a polynucleotide has been altered to encode an amino acidsequence using codons that are preferred in the production cell ororganism, for example, a filamentous fungal cell such as a Trichodermacell. Heterologous nucleotide sequences that are transfected in a hostcell are typically optimized to retain completely or as much as possiblethe amino acid sequence originally encoded by the original (notoptimized) nucleotide sequence. The optimized sequences herein have beenengineered to have codons that are preferred in the correspondingproduction cell or organism, for example the filamentous fungal cell.The amino acid sequences encoded by optimized nucleotide sequences mayalso be referred to as optimized.

As used herein, a “peptide” or a “polypeptide” is an amino acid sequenceincluding a plurality of consecutive polymerized amino acid residues.The peptide or polypeptide may include modified amino acid residues,naturally occurring amino acid residues not encoded by a codon, andnon-naturally occurring amino acid residues. As used herein, a “protein”may refer to a peptide or a polypeptide or a combination of more thanone peptide or polypeptide assembled together by covalent ornon-covalent bonds. Unless specified, the term “protein” may encompassone or more amino acid sequences with their post-translationmodifications, and in particular with either O-mannosylation or N-glycanmodifications.

As used herein, the term “glycoprotein” refers to a protein whichcomprises at least one N-linked glycan attached to at least oneasparagine residue of a protein, or at least one mannose attached to atleast one serine or threonine resulting in O-mannosylation.

The terms “O-mannosylation” or “O-mannosyltransferase activity” are usedherein to refer to the covalent linkage of at least one mannose to onespecific amino acid via one oxygen (typically from serine or threonine).O-mannosyltransferase protein typically adds mannose to hydroxyl groupssuch as hydroxyl of serine or threonine residues.

In particular, O-mannosyltransferase activity may refer to thespecificity of O-mannosyltransferase activity of fungal PMT geneencoding enzymes, and more specifically with the same specificity of T.reesei PMT1.

As used herein, “glycan” refers to an oligosaccharide chain that can belinked to a carrier such as an amino acid, peptide, polypeptide, lipidor a reducing end conjugate. In certain embodiments, the inventionrelates to N-linked glycans (“N-glycan”) conjugated to a polypeptideN-glycosylation site such as -Asn-Xaa-Ser/Thr- by N-linkage toside-chain amide nitrogen of asparagine residue (Asn), where Xaa is anyamino acid residue except Pro. The invention may further relate toglycans as part of dolichol-phospho-oligosaccharide (Dol-P-P-OS)precursor lipid structures, which are precursors of N-linked glycans inthe endoplasmic reticulum of eukaryotic cells. The precursoroligosaccharides are linked from their reducing end to two phosphateresidues on the dolichol lipid. For example, 3-mannosyltransferase Alg3modifies the Do-P-P-oligosaccharide precursor of N-glycans. Generally,the glycan structures described herein are terminal glycan structures,where the non-reducing residues are not modified by other monosaccharideresidue or residues.

As used throughout the present disclosure, glycolipid and carbohydratenomenclature is essentially according to recommendations by theIUPAC-IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res.1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998,257, 29). It is assumed that Gal (galactose), Glc (glucose), GlcNAc(N-acetylglucosamine), GalNAc (N-acetylgalactosamine), Man (mannose),and Neu5Ac are of the D-configuration, Fuc of the L-configuration, andall the monosaccharide units in the pyranose form (D-Galp, D-Glcp,D-GlcpNAc, D-GalpNAc, D-Manp, L-Fucp, D-Neup5Ac). The amine group is asdefined for natural galactose and glucosamines on the 2-position ofGalNAc or GlcNAc. Glycosidic linkages are shown partly in shorter andpartly in longer nomenclature, the linkages of the sialic acidSA/Neu5X-residues α3 and α6 mean the same as α2-3 and α2-6,respectively, and for hexose monosaccharide residues α1-3, α1-6, β1-2,β1-3, β1-4, and β1-6 can be shortened as 3, α6, β2, β3, β4, and β6,respectively. Lactosamine refers to type II N-acetyllactosamine,Galβ4GlcNAc, and/or type I N-acetyllactosamine. Galβ3GlcNAc and sialicacid (SA) refer to N-acetylneuraminic acid (Neu5Ac),N-glycolylneuraminic acid (Neu5Gc), or any other natural sialic acidincluding derivatives of Neu5X. Sialic acid is referred to as NeuNX orNeu5X, where preferably X is Ac or Gc. Occasionally Neu5Ac/Gc/X may bereferred to as NeuNAc/NeuNGc/NeuNX.

The sugars typically constituting N-glycans found in mammalianglycoprotein, include, without limitation, N-acetylglucosamine(abbreviated hereafter as “GlcNAc”), mannose (abbreviated hereafter as“Man”), glucose (abbreviated hereafter as “Glc”), galactose (abbreviatedhereafter as “Gal”), and sialic acid (abbreviated hereafter as“Neu5Ac”). N-glycans share a common pentasaccharide referred to as the“core” structure Man₃GlcNAc₂ (Manα6(Manα3)Manβ4GlcNA4GlcNAc, referred toas Man3). In some embodiments Man3 glycan or its derivativeManα6(GlcNAcβ2Manα3)Man4GlcNA4GlcNAc is the major glycoform. When afucose is attached to the core structure, preferably α6-inked toreducing end GlcNAc, the N-glycan or the core of N-glycan, may berepresented as Man₃GlcNAc₂(Fuc). In an embodiment the major N-glycan isManα3[Manα6(Manα3)Manα6]Man4GlcNA4GlcNAc (Man5).

Preferred hybrid type N-glycans compriseGlcNAcβ2Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (“GlcNAcMan5”), orb4-galactosylated derivatives thereof Galβ4GlcNAcMan3, G1, G2, orGalGlcNAcMan5 glycoform.

A “complex N-glycan” refers to a N-glycan which has at least one GlcNAcresidue, optionally by GlcNAcβ2-residue, on terminal 1,3 mannose arm ofthe core structure and at least one GlcNAc residue, optionally byGlcNAcβ2-residue, on terminal 1,6 mannose arm of the core structure.

Such complex N-glycans include, without limitation, GlcNAc₂Man₃GlcNAc₂(also referred as G0 glycoform), Gal₁GlcNAc₂Man₃GlcNAc₂ (also referredas G1 glycoform), and Gal₂GlcNAc₂Man₃GlcNAc₂ (also referred as G2glycoform), and their core fucosylated glycoforms FG0, FG1 and FG2,respectively GlcNAc₂Man₃GlcNAc₂(Fuc), Gal₁GlcNAc₂Man₃GlcNAc₂(Fuc), andGal₂GlcNAc₂Man₃GlcNAc₂(Fuc).

“Increased” or “Reduced activity of an endogenous enzyme”: Thefilamentous fungal cell may have increased or reduced levels of activityof various endogenous enzymes. A reduced level of activity may beprovided by inhibiting the activity of the endogenous enzyme with aninhibitor, an antibody, or the like. In certain embodiments, thefilamentous fungal cell is genetically modified in ways to increase orreduce activity of various endogenous enzymes. “Genetically modified”refers to any recombinant DNA or RNA method used to create a prokaryoticor eukaryotic host cell that expresses a polypeptide at elevated levels,at lowered levels, or in a mutated form. In other words, the host cellhas been transfected, transformed, or transduced with a recombinantpolynucleotide molecule, and thereby been altered so as to cause thecell to alter expression of a desired protein.

“Genetic modifications” which result in a decrease or deficiency in geneexpression, in the function of the gene, or in the function of the geneproduct (i.e., the protein encoded by the gene) can be referred to asinactivation (complete or partial), knock-out, deletion, disruption,interruption, blockage, silencing, or down-regulation, or attenuation ofexpression of a gene. For example, a genetic modification in a genewhich results in a decrease in the function of the protein encoded bysuch gene, can be the result of a complete deletion of the gene (i.e.,the gene does not exist, and therefore the protein does not exist), amutation in the gene which results in incomplete (disruption) or notranslation of the protein (e.g., the protein is not expressed), or amutation in the gene which decreases or abolishes the natural functionof the protein (e.g., a protein is expressed which has decreased or noenzymatic activity or action). More specifically, reference todecreasing the action of proteins discussed herein generally refers toany genetic modification in the host cell in question, which results indecreased expression and/or functionality (biological activity) of theproteins and includes decreased activity of the proteins (e.g.,decreased catalysis), increased inhibition or degradation of theproteins as well as a reduction or elimination of expression of theproteins. For example, the action or activity of a protein can bedecreased by blocking or reducing the production of the protein,reducing protein action, or inhibiting the action of the protein.Combinations of some of these modifications are also possible. Blockingor reducing the production of a protein can include placing the geneencoding the protein under the control of a promoter that requires thepresence of an inducing compound in the growth medium. By establishingconditions such that the inducer becomes depleted from the medium, theexpression of the gene encoding the protein (and therefore, of proteinsynthesis) could be turned off. Blocking or reducing the action of aprotein could also include using an excision technology approach similarto that described in U.S. Pat. No. 4,743,546. To use this approach, thegene encoding the protein of interest is cloned between specific geneticsequences that allow specific, controlled excision of the gene from thegenome. Excision could be prompted by, for example, a shift in thecultivation temperature of the culture, as in U.S. Pat. No. 4,743,546,or by some other physical or nutritional signal.

In general, according to the present invention, an increase or adecrease in a given characteristic of a mutant or modified protein(e.g., enzyme activity) is made with reference to the samecharacteristic of a parent (i.e., normal, not modified) protein that isderived from the same organism (from the same source or parentsequence), which is measured or established under the same or equivalentconditions. Similarly, an increase or decrease in a characteristic of agenetically modified host cell (e.g., expression and/or biologicalactivity of a protein, or production of a product) is made withreference to the same characteristic of a wild-type host cell of thesame species, and preferably the same strain, under the same orequivalent conditions. Such conditions include the assay or cultureconditions (e.g., medium components, temperature, pH, etc.) under whichthe activity of the protein (e.g., expression or biological activity) orother characteristic of the host cell is measured, as well as the typeof assay used, the host cell that is evaluated, etc. As discussed above,equivalent conditions are conditions (e.g., culture conditions) whichare similar, but not necessarily identical (e.g., some conservativechanges in conditions can be tolerated), and which do not substantiallychange the effect on cell growth or enzyme expression or biologicalactivity as compared to a comparison made under the same conditions.

Preferably, a genetically modified host cell that has a geneticmodification that increases or decreases (reduces) the activity of agiven protein (e.g., an O-mannosyltransferase or protease) has anincrease or decrease, respectively, in the activity or action (e.g.,expression, production and/or biological activity) of the protein, ascompared to the activity of the protein in a parent host cell (whichdoes not have such genetic modification), of at least about 5%, and morepreferably at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55 60%, 65%, 70%, 75 80%, 85 90%, 95%, or any percentage, in wholeintegers between 5% and 100% (e.g., 6%, 7%, 8%, etc.).

In another aspect of the invention, a genetically modified host cellthat has a genetic modification that increases or decreases (reduces)the activity of a given protein (e.g., an O-mannosyltransferase orprotease) has an increase or decrease, respectively, in the activity oraction (e.g., expression, production and/or biological activity) of theprotein, as compared to the activity of the wild-type protein in aparent host cell, of at least about 2-fold, and more preferably at leastabout 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 75-fold,100-fold, 125-fold, 150-fold, or any whole integer increment startingfrom at least about 2-fold (e.g., 3-fold, 4-fold, 5-fold, 6-fold, etc.).

As used herein, the terms “identical” or percent “identity,” in thecontext of two or more nucleic acid or amino acid sequences, refers totwo or more sequences or subsequences that are the same. Two sequencesare “substantially identical” if two sequences have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., 29% identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% or 100% identity over a specified region,or, when not specified, over the entire sequence), when compared andaligned for maximum correspondence over a comparison window, ordesignated region as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection.Optionally, the identity exists over a region that is at least about 50nucleotides (or 10 amino acids) in length, or more preferably over aregion that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200,or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastn, the default parameters are Gap opening penalty=5and Gap extension penalty=2. For blastp, the default parameters are Gapopening penalty=11 and Gap extension penalty=1.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions including, but notlimited to from 20 to 600, usually about 50 to about 200, more usuallyabout 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1981), by the homology alignment algorithm ofNeedleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search forsimilarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA85(8):2444-2448, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection [see, e.g., Brent et al., (2003)Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (RingbouEd)].

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nucleic AcidsRes 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol215(3)-403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. The BLASTN program (for nucleotide sequences) uses asdefaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=−4,and a comparison of both strands. For amino acid sequences, the BLASTPprogram uses as defaults a wordlength of 3, and expectation (E) of 10,and the BLOSUM62 scoring matrix [see Henikoff and Henikoff, (1992) ProcNatl Acad Sci USA 89(22):10915-10919] alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

“Functional variant” or “functional homologous gene” as used hereinrefers to a coding sequence or a protein having sequence similarity witha reference sequence, typically, at least 30%, 40%, 50%, 60%, 70%, 80%,90% or 95% identity with the reference coding sequence or protein, andretaining substantially the same function as said reference codingsequence or protein. A functional variant may retain the same functionbut with reduced or increased activity. Functional variants includenatural variants, for example, homologs from different species orartificial variants, resulting from the introduction of a mutation inthe coding sequence. Functional variant may be a variant with onlyconservatively modified mutations.

“Conservatively modified mutations” as used herein include individualsubstitutions, deletions or additions to an encoded amino acid sequencewhich result in the substitution of an amino acid with a chemicallysimilar amino acid. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. Suchconservatively modified variants are in addition to and do not excludepolymorphic variants, interspecies homologs, and alleles of thedisclosure. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (1), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

Filamentous Fungal Cells

As used herein, “filamentous fungal cells” include cells from allfilamentous forms of the subdivision Eumycota and Oomycota (as definedby Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi,8th edition, 1995, CAB International, University Press, Cambridge, UK).Filamentous fungal cells are generally characterized by a mycelial wallcomposed of chitin, cellulose, glucan, chitosan, mannan, and othercomplex polysaccharides. Vegetative growth is by hyphal elongation andcarbon catabolism is obligately aerobic. In contrast, vegetative growthby yeasts such as Saccharomyces cerevisiae is by budding of aunicellular thallus and carbon catabolism may be fermentative.

Preferably, the filamentous fungal cell is not adversely affected by thetransduction of the necessary nucleic acid sequences, the subsequentexpression of the proteins (e.g., mammalian proteins), or the resultingintermediates. General methods to disrupt genes of and cultivatefilamentous fungal cells are disclosed, for example, for Penicillium, inKopke et al. (2010) Appl Environ Microbiol. 76(14):4664-74. doi:10.1128/AEM.00670-10, for Aspergillus, in Maruyama and Kitamoto (2011),Methods in Molecular Biology, vol. 765, DOI10.1007/978-1-61779-197-0_27;for Neurospora, in Collopy et al. (2010) Methods Mol Biol. 2010;638:33-40. doi: 10.1007/978-1-60761-611-5_3; and for Myceliophthora orChrysosporium PCT/NL2010/000045 and PCT/EP98/06496.

Examples of suitable filamentous fungal cells include, withoutlimitation, cells from an Acremonium, Aspergillus, Fusarium, Humicola,Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, or Trichoderma/Hypocrea strain.

In certain embodiments, the filamentous fungal cell is from aTrichoderma sp., Acremonium, Aspergillus, Aureobasidium, Cryptococcus,Chrysosporium, Chrysosporium lucknowense, Filibasidium, Fusarium,Gibberella, Magnaporthe, Mucor, Myceliophthora, Myrothecium,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Schizophyllum, Talaromyces, Thermoascus, Thielavia, or Tolypocladiumstrain.

In some embodiments, the filamentous fungal cell is a Myceliophthora orChrysosporium, Neurospora or Trichoderma strain.

Aspergillus fungal cells of the present disclosure may include, withoutlimitation, Aspergillus aculeatus, Aspergillus awamori, Aspergillusclavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillusfumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, or Aspergillus terreus.

Neurospora fungal cells of the present disclosure may include, withoutlimitation, Neurospora crassa.

Myceliophthora fungal cells of the present disclosure may include,without limitation, Myceliophthora thermophila.

In a preferred embodiment, the filamentous fungal cell is a Trichodermafungal cell. Trichoderma fungal cells of the present disclosure may bederived from a wild-type Trichoderma strain or a mutant thereof.Examples of suitable Trichoderma fungal cells include, withoutlimitation, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichodermavirens, Trichoderma viride; and alternative sexual form thereof (i.e.,Hypocrea).

In a more preferred embodiment, the filamentous fungal cell is aTrichoderma reesei, and for example, strains derived from ATCC 13631 (QM6a), ATCC 24449 (radiation mutant 207 of QM 6a), ATCC 26921 (QM 9414;mutant of ATCC 24449), VTT-D-00775 (Selinheimo et al., FEBS J., 2006,273: 4322-4335), Rut-C30 (ATCC 56765), RL-P37 (NRRL 15709) or T.harzianum isolate T3 (Wolffhechel, H., 1989).

The invention described herein relates to a PMT deficient filamentousfungal cell, for example selected from Trichoderma, Neurospora,Myceliophthora or a Chrysosporium cells, such as Trichoderma reeseifungal cell, comprising:

-   -   a. at least a first mutation that reduces or eliminates an        endogenous protease activity compared to the parental        filamentous fungal cell which does not have said first mutation        (i.e. a protease-deficient mutation), and,    -   b. at least a second mutation in a PMT gene that reduces or        eliminates an endogenous O-mannosyltransferase activity compared        to a parental filamentous fungal cell which does not have said        second mutation (i.e. a PMT-deficient mutation).        Proteases with Reduced Activity

It has been found that reducing protease activity enables to increasesubstantially the production of heterologous mammalian protein. Indeed,such proteases found in filamentous fungal cells that express aheterologous protein normally catalyse significant degradation of theexpressed recombinant protein. Thus, by reducing the activity ofproteases in filamentous fungal cells that express a heterologousprotein, the stability of the expressed protein is increased, resultingin an increased level of production of the protein, and in somecircumstances, improved quality of the produced protein (e.g.,full-length instead of degraded).

Proteases include, without limitation, aspartic proteases, trypsin-likeserine proteases, subtilisin proteases, glutamic proteases, andsedolisin proteases. Such proteases may be identified and isolated fromfilamentous fungal cells and tested to determine whether reduction intheir activity affects the production of a recombinant polypeptide fromthe filamentous fungal cell. Methods for identifying and isolatingproteases are well known in the art, and include, without limitation,affinity chromatography, zymogram assays, and gel electrophoresis. Anidentified protease may then be tested by deleting the gene encoding theidentified protease from a filamentous fungal cell that expresses arecombinant polypeptide, such a heterologous or mammalian polypeptide,and determining whether the deletion results in a decrease in totalprotease activity of the cell, and an increase in the level ofproduction of the expressed recombinant polypeptide. Methods fordeleting genes, measuring total protease activity, and measuring levelsof produced protein are well known in the art and include the methodsdescribed herein.

Aspartic Proteases

Aspartic proteases are enzymes that use an aspartate residue forhydrolysis of the peptide bonds in polypeptides and proteins. Typically,aspartic proteases contain two highly-conserved aspartate residues intheir active site which are optimally active at acidic pH. Asparticproteases from eukaryotic organisms such as Trichoderma fungi includepepsins, cathepsins, and renins. Such aspartic proteases have atwo-domain structure, which is thought to arise from ancestral geneduplication. Consistent with such a duplication event, the overall foldof each domain is similar, though the sequences of the two domains havebegun to diverge. Each domain contributes one of the catalytic aspartateresidues. The active site is in a cleft formed by the two domains of theaspartic proteases. Eukaryotic aspartic proteases further includeconserved disulfide bridges, which can assist in identification of thepolypeptides as being aspartic acid proteases.

Nine aspartic proteases have been identified in Trichoderma fungalcells: pep1 (tre74156); pep2 (tre53961); pep3 (tre121133); pep4(tre77579), pep5 (tre81004), and pep7 (tre58669), pep8 (tre122076),pep11 (121306), and pep12 (tre119876).

Examples of suitable aspartic proteases include, without limitation,Trichoderma reesei pep1 (SEQ ID NO: 22), Trichoderma reesei pep2 (SEQ IDNO: 18), Trichoderma reesei pep3 (SEQ ID NO: 19); Trichoderma reeseipep4 (SEQ ID NO: 20), Trichoderma reesei pep5 (SEQ ID NO: 21) andTrichoderma reesei pep7 (SEQ ID NO:23), Trichoderma reesei EGR48424 pep8(SEQ ID NO:134), Trichoderma reesei EGR49498 pep11 (SEQ ID NO:135),Trichoderma reesei EGR52517 pep12 (SEQ ID NO:35), and homologs thereof.Examples of homologs of pep1, pep2, pep3, pep4, pep5, pep7, pep8, pep11and pep12 proteases identified in other organisms are also described inPCT/EP/2013/050186, the content of which being incorporated byreference.

Trypsin-Like Serine Proteases

Trypsin-like serine proteases are enzymes with substrate specificitysimilar to that of trypsin. Trypsin-like serine proteases use a serineresidue for hydrolysis of the peptide bonds in polypeptides andproteins. Typically, trypsin-like serine proteases cleave peptide bondsfollowing a positively-charged amino acid residue. Trypsin-like serineproteases from eukaryotic organisms such as Trichoderma fungi includetrypsin 1, trypsin 2, and mesotrypsin. Such trypsin-like serineproteases generally contain a catalytic triad of three amino acidresidues (such as histidine, aspartate, and serine) that form a chargerelay that serves to make the active site serine nucleophilic.Eukaryotic trypsin-like serine proteases further include an “oxyanionhole” formed by the backbone amide hydrogen atoms of glycine and serine,which can assist in identification of the polypeptides as beingtrypsin-like serine proteases.

One trypsin-like serine protease has been identified in Trichodermafungal cells: tsp1 (tre73897). As discussed in PCT/EP/2013/050186, tsp1has been demonstrated to have a significant impact on expression ofrecombinant glycoproteins, such as immunoglobulins.

Examples of suitable tsp1 proteases include, without limitation,Trichoderma reesei tsp1 (SEQ ID NO: 24) and homologs thereof. Examplesof homologs of tsp1 proteases identified in other organisms aredescribed in PCT/EP/2013/050186.

Subtilisin Proteases

Subtilisin proteases are enzymes with substrate specificity similar tothat of subtilisin. Subtilisin proteases use a serine residue forhydrolysis of the peptide bonds in polypeptides and proteins. Generally,subtilisin proteases are serine proteases that contain a catalytic triadof the three amino acids aspartate, histidine, and serine. Thearrangement of these catalytic residues is shared with the prototypicalsubtilisin from Bacillus licheniformis. Subtilisin proteases fromeukaryotic organisms such as Trichoderma fungi include furin, MBTPS1,and TPP2. Eukaryotic trypsin-like serine proteases further include anaspartic acid residue in the oxyanion hole.

Seven subtilisin proteases have been identified in Trichoderma fungalcells: slp1 (tre51365); slp2 (tre123244); slp3 (tre123234); slp5(tre64719), slp6 (tre121495), slp7 (tre123865), and slp8 (tre58698).Subtilisin protease slp7 resembles also sedolisin protease tpp1.

Examples of suitable slp proteases include, without limitation,Trichoderma reesei slp1 (SEQ ID NO: 25), slp2 (SEQ ID NO: 26); slp3 (SEQID NO: 27); slp5 (SEQ ID NO: 28), slp6 (SEQ ID NO: 29), slp7 (SEQ ID NO:30), and slp8 (SEQ ID NO: 31), and homologs thereof. Examples ofhomologs of slp proteases identified in other organisms are described inPCT/EP/2013/050186.

Glutamic Proteases

Glutamic proteases are enzymes that hydrolyse the peptide bonds inpolypeptides and proteins. Glutamic proteases are insensitive topepstatin A, and so are sometimes referred to as pepstatin insensitiveacid proteases. While glutamic proteases were previously grouped withthe aspartic proteases and often jointly referred to as acid proteases,it has been recently found that glutamic proteases have very differentactive site residues than aspartic proteases.

Two glutamic proteases have been identified in Trichoderma fungal cells:gap1 (tre69555) and gap2 (tre106661).

Examples of suitable gap proteases include, without limitation,Trichoderma reesei gap1 (SEQ ID NO: 32), Trichoderma reesei gap2 (SEQ IDNO: 33), and homologs thereof. Examples of homologs of gap proteasesidentified in other organisms are described in PCT/EP/2013/050186.

Sedolisin Proteases and Homologs of Proteases

Sedolisin proteases are enzymes that use a serine residue for hydrolysisof the peptide bonds in polypeptides and proteins. Sedolisin proteasesgenerally contain a unique catalytic triad of serine, glutamate, andaspartate. Sedolisin proteases also contain an aspartate residue in theoxyanion hole. Sedolisin proteases from eukaryotic organisms such asTrichoderma fungi include tripeptidyl peptidase.

Examples of suitable tpp1 proteases include, without limitation,Trichoderma reesei tpp1 tre82623 (SEQ ID NO: 34) and homologs thereof.Examples of homologs of tpp1 proteases identified in other organisms aredescribed in PCT/EP/2013/050186.

As used in reference to protease, the term “homolog” refers to a proteinwhich has protease activity and exhibit sequence similarity with a known(reference) protease sequence. Homologs may be identified by any methodknown in the art, preferably, by using the BLAST tool to compare areference sequence to a single second sequence or fragment of a sequenceor to a database of sequences. As described in the “Definitions”section, BLAST will compare sequences based upon percent identity andsimilarity.

Preferably, a homologous protease has at least 30% identity with(optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 99% or 100% identity over a specified region, or, when notspecified, over the entire sequence), when compared to one of theprotease sequences listed above, including T. reesei pep1, pep2, pep3,pep4, pep5, pep7, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp5,slp6, slp7, slp8, tpp1, gap1 and gap2. Corresponding homologousproteases from N. crassa and M. thermophila are shown in SEQ ID NO:136-169.

Reducing the Activity of Proteases in the Filamentous Fungal Cell of theInvention

The filamentous fungal cells according to the invention have reducedactivity of at least one endogenous protease, typically 2, 3, 4, 5 ormore, in order to improve the stability and production of the proteinwith reduced O-mannosylation in said filamentous fungal cell, preferablyin a PMT-deficient Trichoderma cell.

The activity of proteases found in filamentous fungal cells can bereduced by any method known to those of skill in the art. In someembodiments reduced activity of proteases is achieved by reducing theexpression of the protease, for example, by promoter modification orRNAi.

In further embodiments, the reduced or eliminated expression of theproteases is the result of anti-sense polynucleotides or RNAi constructsthat are specific for each of the genes encoding each of the proteases.In one embodiment, an RNAi construct is specific for a gene encoding anaspartic protease such as a pep-type protease, a trypsin-like serineproteases such as a tsp1, a glutamic protease such as a gap-typeprotease, a subtilisin protease such as a slp-type protease, or asedolisin protease such as a tpp1 or a slp7 protease. In one embodiment,an RNAi construct is specific for the gene encoding a slp-type protease.In one embodiment, an RNAi construct is specific for the gene encodingslp2, slp3, slp5 or slp6. In one embodiment, an RNAi construct isspecific for two or more proteases. In one embodiment, two or moreproteases are any one of the pep-type proteases, any one of thetrypsin-like serine proteasess, any one of the slp-type proteases, anyone of the gap-type proteases and/or any one of the sedolisin proteases.In one embodiment, two or more proteases are slp2, slp3, slp5 and/orslp6. In one embodiment, RNAi construct comprises any one of thefollowing nucleic acid sequences (see also PCT/EP/2013/050186).

RNAi Target sequence GCACACTTTCAAGATTGGC (SEQ ID NO: 15)GTACGGTGTTGCCAAGAAG (SEQ ID NO: 16)GTTGAGTACATCGAGCGCGACAGCATTGTGCACACCATGCTTCCCCTCGAGTCCAAGGACAGCATCATCGTTGAGGACTCGTGCAACGGCGAGACGGAGAAGCAGGCTCCCTGGGGTCTTGCCCGTATCTCTCACCGAGAGACGCTCAACTTTGGCTCCTTCAACAAGTACCTCTACACCGCTGATGGTGGTGAGGGTGTTGATGCCTATGTCATTGACACCGGCACCAACATCGAGCACGTCGACTTTGAGGGTCGTGCCAAGTGGGGCAAGACCATCCCTGCCGGCGATGAGGACGAGGACGGCAACGGCCACGGCACTCACTGCTCTGGTACCGTTGCTGGTAAGAAGTACGGTGTTGCCAAGAAGGCCCACGTCTACGCCGTCAAGGTGCTCCGATCCAACGGATCCGGCACCATGTCTGACGTCGTCAAGGGCGTCGAGTACG (SEQ ID NO: 17)

In other embodiments, reduced activity of proteases is achieved bymodifying the gene encoding the protease. Examples of such modificationsinclude, without limitation, a mutation, such as a deletion ordisruption of the gene encoding said endogenous protease activity.

Accordingly, the invention relates to a filamentous fungal cell, such asa PMT-deficient Trichoderma cell, which has a first mutation thatreduces or eliminates at least one endogenous protease activity comparedto a parental filamentous fungal cell which does not have such proteasedeficient mutation, said filamentous fungal cell further comprising atleast a second mutation in a PMT gene that reduces endogenous proteinO-mannosyltransferase activity compared to a parental Trichoderma cellwhich does not have said second PMT-deficient mutation.

Deletion or disruption mutation includes without limitation knock-outmutation, a truncation mutation, a point mutation, a missense mutation,a substitution mutation, a frameshift mutation, an insertion mutation, aduplication mutation, an amplification mutation, a translocationmutation, or an inversion mutation, and that results in a reduction inthe corresponding protease activity. Methods of generating at least onemutation in a protease encoding gene of interest are well known in theart and include, without limitation, random mutagenesis and screening,site-directed mutagenesis, PCR mutagenesis, insertional mutagenesis,chemical mutagenesis, and irradiation.

In certain embodiments, a portion of the protease encoding gene ismodified, such as the region encoding the catalytic domain, the codingregion, or a control sequence required for expression of the codingregion. Such a control sequence of the gene may be a promoter sequenceor a functional part thereof, i.e., a part that is sufficient foraffecting expression of the gene. For example, a promoter sequence maybe inactivated resulting in no expression or a weaker promoter may besubstituted for the native promoter sequence to reduce expression of thecoding sequence. Other control sequences for possible modificationinclude, without limitation, a leader sequence, a propeptide sequence, asignal sequence, a transcription terminator, and a transcriptionalactivator.

Protease encoding genes that are present in filamentous fungal cells mayalso be modified by utilizing gene deletion techniques to eliminate orreduce expression of the gene. Gene deletion techniques enable thepartial or complete removal of the gene thereby eliminating theirexpression. In such methods, deletion of the gene may be accomplished byhomologous recombination using a plasmid that has been constructed tocontiguously contain the 5′ and 3′ regions flanking the gene.

The protease encoding genes that are present in filamentous fungal cellsmay also be modified by introducing, substituting, and/or removing oneor more nucleotides in the gene, or a control sequence thereof requiredfor the transcription or translation of the gene. For example,nucleotides may be inserted or removed for the introduction of a stopcodon, the removal of the start codon, or a frame-shift of the openreading frame. Such a modification may be accomplished by methods knownin the art, including without limitation, site-directed mutagenesis andpeR generated mutagenesis (see, for example, Botstein and Shortie, 1985,Science 229: 4719; Lo et al., 1985, Proceedings of the National Academyof Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids Research16: 7351; Shimada, 1996, Meth. Mol. Bioi. 57: 157; Ho et al., 1989, Gene77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar and Sommer, 1990,BioTechniques 8: 404).

Additionally, protease encoding genes that are present in filamentousfungal cells may be modified by gene disruption techniques by insertinginto the gene a disruptive nucleic acid construct containing a nucleicacid fragment homologous to the gene that will create a duplication ofthe region of homology and incorporate construct nucleic acid betweenthe duplicated regions. Such a gene disruption can eliminate geneexpression if the inserted construct separates the promoter of the genefrom the coding region or interrupts the coding sequence such that anonfunctional gene product results. A disrupting construct may be simplya selectable marker gene accompanied by 5′ and 3′ regions homologous tothe gene. The selectable marker enables identification of transformantscontaining the disrupted gene.

Protease encoding genes that are present in filamentous fungal cells mayalso be modified by the process of gene conversion (see, for example,Iglesias and Trautner, 1983, Molecular General Genetics 189:5 73-76).For example, in the gene conversion a nucleotide sequence correspondingto the gene is mutagenized in vitro to produce a defective nucleotidesequence, which is then transformed into a Trichoderma strain to producea defective gene. By homologous recombination, the defective nucleotidesequence replaces the endogenous gene. It may be desirable that thedefective nucleotide sequence also contains a marker for selection oftransformants containing the defective gene.

Protease encoding genes of the present disclosure that are present infilamentous fungal cells that express a recombinant polypeptide may alsobe modified by established anti-sense techniques using a nucleotidesequence complementary to the nucleotide sequence of the gene (see, forexample, Parish and Stoker, 1997, FEMS Microbiology Letters 154:151-157). In particular, expression of the gene by filamentous fungalcells may be reduced or inactivated by introducing a nucleotide sequencecomplementary to the nucleotide sequence of the gene, which may betranscribed in the strain and is capable of hybridizing to the mRNAproduced in the cells. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the mRNA, the amount ofprotein translated is thus reduced or eliminated.

Protease encoding genes that are present in filamentous fungal cells mayalso be modified by random or specific mutagenesis using methods wellknown in the art, including without limitation, chemical mutagenesis(see, for example, Hopwood, The Isolation of Mutants in Methods inMicrobiology (J. R. Norris and D. W. Ribbons, eds.) pp. 363-433,Academic Press, New York, 25 1970). Modification of the gene may beperformed by subjecting filamentous fungal cells to mutagenesis andscreening for mutant cells in which expression of the gene has beenreduced or inactivated. The mutagenesis, which may be specific orrandom, may be performed, for example, by use of a suitable physical orchemical mutagenizing agent, use of a suitable oligonucleotide,subjecting the DNA sequence to peR generated mutagenesis, or anycombination thereof. Examples of physical and chemical mutagenizingagents include, without limitation, ultraviolet (UV) irradiation,hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the filamentous fungal cells, such asTrichoderma cells, to be mutagenized in the presence of the mutagenizingagent of choice under suitable conditions, and then selecting formutants exhibiting reduced or no expression of the gene.

In certain embodiments, the at least one mutation or modification in aprotease encoding gene of the present disclosure results in a modifiedprotease that has no detectable protease activity. In other embodiments,the at least one modification in a protease encoding gene of the presentdisclosure results in a modified protease that has at least 25% less, atleast 50% less, at least 75% less, at least 90%, at least 95%, or ahigher percentage less protease activity compared to a correspondingnon-modified protease.

The filamentous fungal cells or Trichoderma fungal cells of the presentdisclosure may have reduced or no detectable protease activity of atleast three, or at least four proteases selected from the groupconsisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1,slp1, slp2, slp3, slp5, slp6, slp7, gap1 and gap2. In preferredembodiment, a filamentous fungal cell according to the invention is aPMT-deficient filamentous fungal cell which has a deletion or disruptionin at least 3 or 4 endogenous proteases, resulting in no detectableactivity for such deleted or disrupted endogenous proteases and furthercomprising another mutation in a PMT gene that reduces endogenousprotein O-mannosyltransferase activity compared to a parentalTrichoderma cell which does not have said mutation.

In certain embodiments, the PMT-deficient filamentous fungal cell orTrichoderma cell, has reduced or no detectable protease activity inpep1, tsp1, and slp1. In other embodiments, the PMT-deficientfilamentous fungal cell or Trichoderma cell, has reduced or nodetectable protease activity in gap1, slp1, and pep1. In certainembodiments, the PMT-deficient filamentous fungal cell or Trichodermacell, has reduced or no detectable protease activity in slp2, pep1 andgap1. In certain embodiments, the PMT-deficient filamentous fungal cellor Trichoderma cell, has reduced or no detectable protease activity inslp2, pep1, gap1 and pep4. In certain embodiments, the PMT-deficientfilamentous fungal cell or Trichoderma cell, has reduced or nodetectable protease activity in slp2, pep1, gap1, pep4 and slp1. Incertain embodiments, the PMT-deficient filamentous fungal cell orTrichoderma cell, has reduced or no detectable protease activity inslp2, pep1, gap1, pep4, slp1, and slp3. In certain embodiments, thePMT-deficient filamentous fungal cell or Trichoderma cell, has reducedor no detectable protease activity in slp2, pep1, gap1, pep4, slp1,slp3, and pep3. In certain embodiments, the PMT-deficient filamentousfungal cell or Trichoderma cell, has reduced or no detectable proteaseactivity in slp2, pep1, gap1, pep4, slp1, slp3, pep3 and pep2. Incertain embodiments, the PMT-deficient filamentous fungal cell orTrichoderma cell, has reduced or no detectable protease activity inslp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2 and pep5. In certainembodiments, the PMT-deficient filamentous fungal cell or Trichodermacell, has reduced or no detectable protease activity in slp2, pep1,gap1, pep4, slp1, slp3, pep3, pep2, pep5 and tsp1. In certainembodiments, the PMT-deficient filamentous fungal cell or Trichodermacell, has reduced or no detectable protease activity in slp2, pep1,gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1 and slp7. In certainembodiments, the PMT-deficient filamentous fungal cell or Trichodermacell, has reduced or no detectable protease activity in slp2, pep1,gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1, slp7 and slp8. Incertain embodiments, the PMT-deficient filamentous fungal cell orTrichoderma cell, has reduced or no detectable protease activity inslp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5, tsp1, slp7, slp8and gap2. In certain embodiments, the PMT-deficient filamentous fungalcell or Trichoderma cell, has reduced or no detectable protease activityin at least three endogenous proteases selected from the groupconsisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1,slp2, slp3, slp7, gap1 and gap2. In certain embodiments, thePMT-deficient filamentous fungal cell or Trichoderma cell, has reducedor no detectable protease activity in at least three to six endogenousproteases selected from the group consisting of pep1, pep2, pep3, pep4,pep5, tsp1, slp1, slp2, slp3, gap1 and gap2. In certain embodiments, thePMT-deficient filamentous fungal cell or Trichoderma cell, has reducedor no detectable protease activity in at least seven to ten endogenousproteases selected from the group consisting of pep1, pep2, pep3, pep4,pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1,gap1 and gap2.

In one embodiment that may be combined with the precedent embodiments,the filamentous fungal cell of the invention does not comprise adeletion or disruption of an endogenous gene encoding a chaperoneprotein. In particular, said filamentous fungal cell of the inventionexpresses functional endogenous chaperone protein Protein DisulphideIsomerase (PDI).

Endogenous O-Mannosyltransferase in Filamentous Fungal Cells

O-mannosyltransferases are encoded by pmt genes in yeasts andfilamentous fungi, which can be divided into three subfamilies, based onsequence homologies: PMT1, PMT2 and PMT4.

For example, in yeast S. cerevisiae, 7 different PMTs have beencharacterized: ScPMT1, ScPMT5 and ScPMT7 belong to the PMT1 subfamily.ScPMT2, ScPMT3 and ScPMT6 belong to the PMT2 subfamily and ScPMT4belongs to the PMT4 subfamily. Such O-mannosyltransferases and theircoding sequences may be identified and isolated from filamentous fungalcells and tested to determine whether reduction in their activityenables the reduction of O-mannosylation on secreted O-mannosylatedrecombinant protein preferably not affecting the production of suchrecombinant polypeptide from the filamentous fungal cell. Methods foridentifying and isolating PMTs are well known in the art. An identifiedO-mannosyltransferase may then be tested by deleting the gene encodingthe identified O-mannosyltransferase from a filamentous fungal cell thatexpresses a recombinant O-mannosylated protein, such a heterologous ormammalian O-mannosylated protein, and determining whether the deletionresults in a decrease in total O-mannosyltransferase activity of thecell, preferably not affecting the level of production of the expressedrecombinant protein. Methods for deleting genes and measuring levels ofproduced protein are well known in the art and include the methodsdescribed herein.

Three O-mannosyltransferases have been identified in Trichoderma fungalcells: pmt1, pmt2 and pmt3, belonging respectively based on sequencehomologies to the PMT4, PMT1 and PMT2 subfamily.

Examples of suitable O-mannosyltransferase include, without limitation,Trichoderma reesei pmt1 (SEQ ID NO: 2), Trichoderma reesei pmt2 (SEQ IDNO: 3), Trichoderma reesei pmt3 (SEQ ID NO: 4) and homologs thereof.FIG. 5 shows phylogeny of pmt homologs in selected filamentous fungi andFIG. 6 shows an alignment of pmt1 conserved domains among differentspecies.

In a preferred embodiment, said PMT-deficient filamentous fungal cell,e.g., a Trichoderma cell, has at least one mutation in a PMT geneselected from the group consisting of:

-   -   a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1,    -   b) a functional homologous gene of PMT1 gene, which functional        homologous gene is capable of restoring parental O-mannosylation        level by functional complementation when introduced into a T.        reesei strain having a disruption in said PMT1 gene, and,    -   c) a polynucleotide encoding a polypeptide having at least 50%,        at least 60%, at least 70%, at least 90%, or at least 95%        identity with SEQ ID NO:2, said polypeptide having protein        O-mannosyltransferase activity.

More preferably, said PMT-deficient filamentous fungal cell, e.g., aTrichoderma cell, has at least one mutation in a PMT gene which

-   -   a) has a polynucleotide encoding a polypeptide having at least        50%, at least 60%, at least 70%, at least 90%, or at least 95%        identity with SEQ ID NO:2, and,    -   b) is capable of restoring, at least 50%, preferably about 100%        of parental O-mannosylation level by functional complementation        when introduced into a T. reesei strain having a disruption in        a T. reesei PMT1 gene.

Methods for disrupting PMT1 gene in T. reesei are disclosed in theExamples below.

Sequences of homologs of pmt1 in filamentous fungi can be found in thedatabases using sequence alignment search tools, such as BLASTalgorithm. It includes without limitation, A. oryzae gi391865791,EIT75070.1 (SEQ ID NO:5), A. niger gi317036343, XP_001398147.2 (SEQ IDNO:6), A. nidulans gi67522004, XP_659063.1 (SEQ ID NO:7), T. virensgi358379774, EHK17453.1 (SEQ ID NO:8), T. atroviride gi358400594,EHK49920.1 (SEQ ID NO:9), F. oxysporum gi342879728, EGU80965.1 (SEQ IDNO:10), G. zeae gi46107450, XP_380784.1 (SEQ ID NO:11), M. thermophilagi367020262, XP_003659416.1 (SEQ ID NO:12), N. crassa gi164423013,XP_963926.2 (SEQ ID NO:13), and P. chrysogenum gi255953619,XP_002567562.1 (SEQ ID NO:14).

Reducing Endogenous Protein O-Mannosyltransferase Activity inFilamentous Fungal Cell of the Invention

The PMT-deficient filamentous fungal cells according to the inventionhave reduced activity of at least one O-mannosyltransferase activity, inorder to reduce or decrease O-mannosylation in said filamentous fungalcell, preferably Trichoderma cell.

The activity of said O-mannosyltransferases found in filamentous fungalcells can be reduced by any method known to those of skill in the art.In some embodiments reduced activity of O-mannosyltransferases isachieved by reducing the expression of the O-mannosyltransferases, forexample, by promoter modification or RNAi.

In other embodiments, reduced activity of O-mannosyltransferases isachieved by modifying the gene encoding the O-mannosyltransferase.Examples of such modifications include, without limitation, a mutation,such as a deletion or disruption of the gene encoding said endogenousO-mannosyltransferase activity.

Deletion or disruption mutation can be performed as described in theabove sections, in particular in relation to deletion or disruption ofgenes encoding proteases. These includes without limitation knock-outmutation, a truncation mutation, a point mutation, a missense mutation,a substitution mutation, a frameshift mutation, an insertion mutation, aduplication mutation, an amplification mutation, a translocationmutation, or an inversion mutation, and that results in a reduction inthe corresponding O-mannosyltransferase activity.

In certain embodiments, the mutation or modification in anO-mannosyltransferase (PMT) encoding gene of the present disclosureresults in a modified O-mannosyltransferase that has no detectableO-mannosyltransferase activity. In other embodiments, the at least onemodification in a O-mannosyltransferase encoding gene of the presentdisclosure results in a modified O-mannosyltransferase that has at least25% less, at least 50% less, at least 75% less, at least 90%, at least95%, or a higher percentage less O-mannosyltransferase activity comparedto a corresponding non-modified O-mannosyltransferase.

In preferred embodiment, a mutation that reduces endogenous proteinO-mannosyltransferase activity in a PMT-deficient filamentous fungalcell, e.g. Trichoderma cell, is a PMT-deficient cell which has adeletion or disruption of a PMT gene encoding said O-mannosyltransferaseactivity, resulting in no detectable expression for such deleted ordisrupted PMT gene.

One specific embodiment of the present invention is a PMT-deficientTrichoderma reesei cell, comprising

-   -   a. at least a first mutation that reduces an endogenous protease        activity compared to a parental Trichoderma cell which does not        have said first mutation, and,    -   b. at least a disruption or deletion of PMT1 gene of T. reesei.    -   c. optionally, said cell further express a heterologous protein        with serine or threonine, which has reduced O-mannosylation due        to said mutation in said PMT gene.

The reduction (or decrease) of O-mannosyltransferase activity may bedetermined by comparing the O-mannosylation level of a heterologousprotein in PMT-deficient filamentous fungal cell according to theinvention, with the O-mannosylation level of a heterologous protein inthe parental cell which does not have said PMT-deficient mutation.

In specific embodiments, the PMT-deficient filamentous fungal cellaccording to the invention expresses a heterologous protein which hasreduced O-mannosylation due to said mutation in said PMT gene and theO-mannosylation level on the expressed heterologous protein is at least20%, 40%, 50%, 60%, 70%, 80%, or 90% lower than the O-mannosylationlevel of the heterologous protein when expressed in the parentalfilamentous fungal cell which does not have said second PMT-deficientmutation.

O-mannosylation level may also be determined as mole % of O-mannosylatedpolypeptide per total polypeptide as produced by the host cell of theinvention. Analytical methods, such as MALDI TOF MS analysis may be usedto determine O-mannosylation level as described in detail in the Example1 below, section entitled “Analyses of Dpmt1 strains M403, M404, M406and M407. In brief, a polypeptide as produced by the PMT-deficientfilamentous fungal cell is purified to determine its O-mannoslyationlevel. Non O-mannosylated, and O-mannosylated structure of thepolypeptide are separated and quantified by MALDI-TOF MS analysis. Forexample, the quantification of O-mannosylation level may be performed bydetermining area values or intensity of the different peaks of MALDI-TOFMS spectrum. An O-mannosylation level of 5% as determined by suchmethod, using area values or intensity, reflects that about 95% (mol %)of the analysed polypeptides in the composition are not O-mannosylatedlnspecific embodiments, the PMT-deficient filamentous fungal cellexpresses a heterologous protein which has reduced O-mannosylation dueto said mutation in said PMT gene, and the O-mannosylation level on theexpressed heterologous protein (for example, as defined above bydetermining area or intensity values of MALDI TOF MS spectrum peaks) isreduced to less than 25%, 20%, 17%, 15%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1%, or 0.5% (as mole % of mannose residues perpolypeptide chain).

In an embodiment, the heterologous protein with reduced O-mannosylationis selected from the group consisting of:

-   -   a) an immunoglubulin, such as IgG,    -   b) a light chain or heavy chain of an immunoglobulin,    -   c) a heavy chain or alight chain of an antibody,    -   d) a single chain antibody,    -   e) a camelid antibody,    -   f) a monomeric or multimeric single domain antibody,    -   g) a FAb-fragment, a FAb2-fragment, and,    -   h) their antigen-binding fragments.

In a specific embodiment, a mutation that reduces endogenousO-mannosyltransferase activity is a deletion or a disruption of a PMTgene encoding said engogenous protein O-mannosyltransferase activity.For example in Trichoderma cell, a mutation that reduces endogenousO-mannosyltransferase activity is a deletion or a disruption of a PMT1gene.

Filamentous Fungal Cell for Producing Glycoproteins with ReducedO-Mannosylation and Mammalian-Like N-Glycans

The filamentous fungal cells according to the present invention may beuseful in particular for producing heterologous glycoproteins withreduced O-mannosylation and mammalian-like N-glycans, such as complexN-glycans.

Accordingly, in one aspect, the filamentous fungal cell is furthergenetically modified to produce a mammalian-like N-glycan, therebyenabling in vivo production of glycoprotein with no or reducedO-mannosylation and with mammalian-like N-glycan as major glycoforms.

In certain embodiments, this aspect includes methods of producingglycoproteins with mammalian-like N-glycans in a Trichoderma cell.

In certain embodiment, the glycoprotein comprises, as a major glycoform,the mammalian-like N-glycan having the formula[(Galβ4)_(x)GlcNAcβ2]_(z)Manα3([(Galβ4)_(y)GlcNAcβ2]_(w)Manα6)Man{β4GlcNAcβGlcNAc,where ( ) defines a branch in the structure, where [ ] or { } define apart of the glycan structure either present or absent in a linearsequence, and where x, y, z and w are 0 or 1, independently. In anembodiment w and z are 1.

In certain embodiments, the glycoprotein comprises, as a majorglycoform, mammalian-like N-glycan selected from the group consistingof:

-   -   i. Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (Man5 glycoform);    -   ii. GlcNAcβ2Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc        (GlcNAcMan5 glycoform);    -   iii. Manα6(Manα3)Manβ4GlcNAβ4GlcNAc (Man3 glycoform);    -   iv. Manα6(GlcNAcβ2Manα3)Manβ4GlcNAβ4GlcNAc (GlcNAcMan3) or,    -   v. complex type N-glycans selected from the G0, G1, or G2        glycoform.

In an embodiment, the glycoprotein composition with mammalian-likeN-glycans, preferably produced by an alg3 knock-out strain, includeglycoforms that essentially lack or are devoid of glycansManα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (Man5). In specificembodiments, the filamentous fungal cell produces glycoproteins with, asmajor glycoform, the trimannosyl N-glycan structureManα3[Manα6]Manβ4GlcNAcβ4GlcNAc. In other embodiments, the filamentousfungal cell procudes glycoproteins with, as major glycoform, the G0N-glycan structure GlcNAcβ2Manα3[GlcNAcβ2Manα6]Manβ4GlcNAcβ4GlcNAc.

In certain embodiments, the PMT-deficient filamentous fungal cell of theinvention produces glycoprotein composition with a mixture of differentN-glycans.

In some embodiments, Man3GlcNAc2 N-glycan (i.e.Manα3[Manα6]Manβ4GlcNAcβ4GlcNAc) represents at least 10%, at least 20%,at least at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90% or more of total (mol %) neutralN-glycans of a heterologous protein with reduced O-mannosylation, asexpressed in a filamentous fungal cells of the invention.

In other embodiments, GlcNAc2Man3 N-glycan (for example G0GlcNAcβ2Manα3[GlcNAcβ2Manα6]Manβ4GlcNAcβ4GlcNAc) represents at least10%, at least 20%, at least at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90% or more of total(mol %) neutral N-glycans of a heterologous protein with reducedO-mannosylation, as expressed in a filamentous fungal cells of theinvention.

In other embodiments, GalGlcNAc2Man3GlcNAc2 N-glycan (for example G1N-glycan) represents at least 10%, at least 20%, at least at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or more of total (mol %) neutral N-glycans of a heterologousprotein with reduced O-mannosylation, as expressed in a filamentousfungal cells of the invention.

In other embodiments, Gal2GlcNAc2Man3GlcNAc2 N-glycan (for example G2N-glycan) represents at least 10%, at least 20%, at least at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90% or more of total (mol %) neutral N-glycans of a heterologousprotein with reduced O-mannosylation, as expressed in a filamentousfungal cells of the invention.

In other embodiments, complex type N-glycan represents at least 10%, atleast 20%, at least at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or more of total (mol %)neutral N-glycans of a heterologous protein with reducedO-mannosylation, as expressed in a filamentous fungal cells of theinvention.

In other embodiments, hybrid type N-glycan represents at least 10%, atleast 20%, at least at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90% or more of total (mol %)neutral N-glycans of a heterologous protein with reducedO-mannosylation, as expressed in a filamentous fungal cells of theinvention.

In other embodiments, less than 0.5%, 0.1%, 0.05%, or less than 0.01% ofthe N-glycan of the glycoprotein composition produced by the host cellof the invention, comprises galactose. In certain embodiments, none ofN-glycans comprise galactose.

The Neu5Gc and Galα− (non-reducing end terminal Galα3Galβ4GlcNAc)structures are known xenoantigenic (animal derived) modifications ofantibodies which are produced in animal cells such as CHO cells. Thestructures may be antigenic and, thus, harmful even at lowconcentrations. The filamentous fungi of the present invention lackbiosynthetic pathways to produce the terminal Neu5Gc and Galα−structures. In an embodiment that may be combined with the precedingembodiments less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/orO-glycans of the glycoprotein composition comprises Neu5Gc and/or Galα−structure. In an embodiment that may be combined with the precedingembodiments, less than 0.1%, 0.01%, 0.001% or 0% of the N-glycans and/orO-glycans of the antibody composition comprises Neu5Gc and/or Galα−structure.

The filamentous fungal cells of the present invention lack genes toproduce fucosylated heterologous proteins. In an embodiment that may becombined with the preceding embodiments, less than 0.1%, 0.01%, 0.001%,or 0% of the N-glycan of the glycoprotein composition comprises corefucose structures. In an embodiment that may be combined with thepreceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of theN-glycan of the antibody composition comprises core fucose structures.

The terminal Galβ4GlcNAc structure of N-glycan of mammalian cellproduced glycans affects bioactivity of antibodies and Galβ3GlcNAc maybe xenoantigen structure from plant cell produced proteins. In anembodiment that may be combined with one or more of the precedingembodiments, less than 0.1%, 0.01%, 0.001%, or 0% of N-glycan of theglycoprotein composition comprises terminal galactose epitopesGalβ3/4GlcNAc. In an embodiment that may be combined with one or more ofthe preceding embodiments, less than 0.1%, 0.01%, 0.001%, or 0% of theN-glycan of the antibody composition comprises terminal galactoseepitopes Galβ3/4GlcNAc.

Glycation is a common post-translational modification of proteins,resulting from the chemical reaction between reducing sugars such asglucose and the primary amino groups on protein. Glycation occurstypically in neutral or slightly alkaline pH in cell culturesconditions, for example, when producing antibodies in CHO cells andanalysing them (see, for example, Zhang et al. (2008) Unveiling aglycation hot spot in a recombinant humanized monoclonal antibody. AnalChem. 80(7):2379-2390). As filamentous fungi of the present inventionare typically cultured in acidic pH, occurrence of glycation is reduced.In an embodiment that may be combined with the preceding embodiments,less than 1.0%, 0.5%, 0.1%, 0.01%, 0.001%, or 0% of the glycoproteincomposition comprises glycation structures. In an embodiment that may becombined with the preceding embodiments, less than 1.0%, 0.5%, 0.1%,0.01%, 0.001%, or 0% of the antibody composition comprises glycationstructures.

In one embodiment, the glycoprotein composition, such as an antibody isdevoid of one, two, three, four, five, or six of the structures selectedfrom the group of Neu5Gc, terminal Galα3Galβ4GlcNAc, terminalGalβ4GlcNAc, terminal Galβ3GlcNAc, core linked fucose and glycationstructures.

In certain embodiments, such glycoprotein protein with mammalian-likeN-glycan and reduced O-mannosylation, as produced in the filamentousfungal cell of the invention, is a therapeutic protein. Therapeuticproteins may include immunoglobulin, or a protein fusion comprising a Fcfragment or other therapeutic glycoproteins, such as antibodies,erythropoietins, interferons, growth hormones, albumins or serumalbumin, enzymes, or blood-clotting factors and may be useful in thetreatment of humans or animals. For example, the glycoproteins withmammalian-like N-glycan and reduced O-mannosylation as produced by thefilamentous fungal cell according to the invention may be a therapeuticglycoprotein such as rituximab.

Methods for producing glycoproteins with mammalian-like N-glycans infilamentous fungal cells are also described for example inWO2012/069593.

In one aspect, the filamentous fungal cell according to the invention asdescribed above, is further genetically modified to mimick thetraditional pathway of mammalian cells, starting from Man5 N-glycans asacceptor substrate for GnTI, and followed sequentially by GnT1,mannosidase II and GnTII reaction steps (hereafter referred as the“traditional pathway” for producing G0 glycoforms). In one variant, asingle recombinant enzyme comprising the catalytic domains of GnTI andGnTII, is used.

Alternatively, in a second aspect, the filamentous fungal cell accordingto the invention as described above is further genetically modified tohave alg3 reduced expression, allowing the production of coreMan₃GlcNAc₂ N-glycans, as acceptor substrate for GnTI and GnTIIsubsequent reactions and bypassing the need for mannosidase α1,2 ormannosidase II enzymes (the reduced “alg3” pathway). In one variant, asingle recombinant enzyme comprising the catalytic domains of GnTI andGnTII, is used.

In such embodiments for mimicking the traditional pathway for producingglycoproteins with mammalian-like N-glycans, a Man₅ expressingfilamentous fungal cell, such as T. reesei strain, may be transformedwith a GnTI or a GnTII/GnTI fusion enzyme using random integration or bytargeted integration to a known site known not to affect Man5glycosylation. Strains that synthesise GlcNAcMan5 N-glycan forproduction of proteins having hybrid type glycan(s) are selected. Theselected strains are further transformed with a catalytic domain of αmannosidase II-type mannosidase capable of cleaving Man5 structures togenerate GlcNAcMan3 for production of proteins having the correspondingGlcNAcMan3 glycoform or their derivative(s). In certain embodiments,mannosidase II-type enzymes belong to glycoside hydrolase family 38(cazy.org/GH38_all.html). Characterized enzymes include enzymes listedin cazy.org/GH38_characterized.html. Especially useful enzymes areGolgi-type enzymes that cleaving glycoproteins, such as those ofsubfamily α-mannosidase II (Man2A1;ManA2). Examples of such enzymesinclude human enzyme AAC50302, D. melanogaster enzyme (Van den Elsen J.M. et al (2001) EMBO J. 20: 3008-3017), those with the 3D structureaccording to PDB-reference 1HTY, and others referenced with thecatalytic domain in PDB. For cytoplasmic expression, the catalyticdomain of the mannosidase is typically fused with an N-terminaltargeting peptide (for example as disclosed in the above Section) orexpressed with endogenous animal or plant Golgi targeting structures ofanimal or plant mannosidase II enzymes. After transformation with thecatalytic domain of α mannosidase II-type mannosidase, strains areselected that produce GlcNAcMan3 (if GnTI is expressed) or strains areselected that effectively produce GlcNAc2Man3 (if a fusion of GnTI andGnTII is expressed). For strains producing GlcNAcMan3, such strains arefurther transformed with a polynucleotide encoding a catalytic domain ofGnTII and transformant strains that are capable of producingGlcNAc2Man3GlcNAc2 are selected.

In such embodiment for mimicking the traditional pathway, thefilamentous fungal cell is a PMT-deficient filamentous fungal cell asdefined in previous sections, and further comprising one or morepolynucleotides encoding a polypeptide selected from the groupconsisting of:

-   -   i) α1,2 mannosidase,    -   ii) N-acetylglucosaminyltransferase I catalytic domain,    -   iii) α mannosidase II,    -   iv) N-acetylglucosaminyltransferase II catalytic domain,    -   v) β1,4 galactosyltransferase, and,    -   vi) fucosyltransferase.

In embodiments using the reduced alg3 pathway, the filamentous fungalcell, such as a Trichoderma cell, has a reduced level of activity of adolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase comparedto the level of activity in a parent host cell.Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (EC2.4.1.130) transfers an alpha-D-mannosyl residue from dolichyl-phosphateD-mannose into a membrane lipid-linked oligosaccharide. Typically, thedolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase enzyme isencoded by an alg3 gene. In certain embodiments, the filamentous fungalcell for producing glycoproteins with mammalian-like N-glycans has areduced level of expression of an alg3 gene compared to the level ofexpression in a parent strain.

More preferably, the filamentous fungal cell comprises a mutation ofalg3. The ALG3 gene may be mutated by any means known in the art, suchas point mutations or deletion of the entire alg3 gene. For example, thefunction of the alg3 protein is reduced or eliminated by the mutation ofalg3. In certain embodiments, the alg3 gene is disrupted or deleted fromthe filamentous fungal cell, such as Trichoderma cell. In certainembodiments, the filamentous fungal cell is a T. reesei cell. SEQ IDNOs: 36 and 37 provide, the nucleic acid and amino acid sequences of thealg3 gene in T. reesei, respectively. In an embodiment the filamentousfungal cell is used for the production of a glycoprotein, wherein theglycan(s) comprise or consist of Manα3[Manα6]Manβ4GlcNAcβ4GlcNAc, and/ora non-reducing end elongated variant thereof.

In certain embodiments, the filamentous fungal cell has a reduced levelof activity of an alpha-1,6-mannosyltransferase compared to the level ofactivity in a parent strain. Alpha-1,6-mannosyltransferase (EC2.4.1.232) transfers an alpha-D-mannosyl residue from GDP-mannose into aprotein-linked oligosaccharide, forming an elongation initiatingalpha-(1->6)-D-mannosyl-D-mannose linkage in the Golgi apparatus.Typically, the alpha-1,6-mannosyltransferase enzyme is encoded by anoch1 gene. In certain embodiments, the filamentous fungal cell has areduced level of expression of an och1 gene compared to the level ofexpression in a parent filamentous fungal cell. In certain embodiments,the och1 gene is deleted from the filamentous fungal cell.

The filamentous fungal cells used in the methods of producingglycoprotein with mammalian-like N-glycans may further contain apolynucleotide encoding an N-acetylglucosaminyltransferase I catalyticdomain (GnTI) that catalyzes the transfer of N-acetylglucosamine to aterminal Manα3 and a polynucleotide encoding anN-acetylglucosaminyltransferase II catalytic domain (GnTII), thatcatalyses N-acetylglucosamine to a terminal Manα6 residue of an acceptorglycan to produce a complex N-glycan. In one embodiment, saidpolynucleotides encoding GnTI and GnTII are linked so as to produce asingle protein fusion comprising both catalytic domains of GnTI andGnTII.

As disclosed herein, N-acetylglucosaminyltransferase I (GlcNAc-TI; GnTI;EC 2.4.1.101) catalyzes the reactionUDP-N-acetyl-D-glucosamine+3-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>UDP+3-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl-R,where R represents the remainder of the N-linked oligosaccharide in theglycan acceptor. An N-acetylglucosaminyltransferase I catalytic domainis any portion of an N-acetylglucosaminyltransferase I enzyme that iscapable of catalyzing this reaction. GnTI enzymes are listed in the CAZydatabase in the glycosyltransferase family 13 (cazy.org/GT13_all).Enzymatically characterized species includes A. thaliana AAR78757.1(U.S. Pat. No. 6,653,459), C. elegans AAD03023.1 (Chen S. et al J. Biol.Chem 1999; 274(1):288-97), D. melanogaster AAF57454.1 (Sarkar &Schachter Biol Chem. 2001 February; 382(2):209-17); C. griseusAAC52872.1 (Puthalakath H. et al J. Biol. Chem 1996 271(44):27818-22);H. sapiens AAA52563.1 (Kumar R. et al Proc Natl Acad Sci USA. 1990December; 87(24):9948-52); M. auratus AAD04130.1 (Opat As et al BiochemJ. 1998 Dec. 15; 336 (Pt 3):593-8), (including an example ofdeactivating mutant), Rabbit, O. cuniculus AAA31493.1 (Sarkar M et al.Proc Natl Acad Sci USA. 1991 Jan. 1; 88(1):234-8). Amino acid sequencesfor N-acetylglucosaminyltransferase I enzymes from various organisms aredescribed for example in PCT/EP2011/070956. Additional examples ofcharacterized active enzymes can be found atcazy.org/GT13_characterized. The 3D structure of the catalytic domain ofrabbit GnTI was defined by X-ray crystallography in Unligil U M et al.EMBO J. 2000 Oct. 16; 19(20):5269-80. The Protein Data Bank (PDB)structures for GnTI are 1FO8, 1FO9, 1FOA, 2AM3, 2AM4, 2AM5, and 2APC. Incertain embodiments, the N-acetylglucosaminyltransferase I catalyticdomain is from the human N-acetylglucosaminyltransferase I enzyme (SEQID NO: 38) or variants thereof. In certain embodiments, theN-acetylglucosaminyltransferase I catalytic domain contains a sequencethat is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to amino acid residues 84-445 of SEQ ID NO: 38.In some embodiments, a shorter sequence can be used as a catalyticdomain (e.g. amino acid residues 105-445 of the human enzyme or aminoacid residues 107-447 of the rabbit enzyme; Sarkar et al. (1998)Glycoconjugate J 15:193-197). Additional sequences that can be used asthe GnTI catalytic domain include amino acid residues from about aminoacid 30 to 445 of the human enzyme or any C-terminal stem domainstarting between amino acid residue 30 to 105 and continuing to aboutamino acid 445 of the human enzyme, or corresponding homologous sequenceof another GnTI or a catalytically active variant or mutant thereof. Thecatalytic domain may include N-terminal parts of the enzyme such as allor part of the stem domain, the transmembrane domain, or the cytoplasmicdomain.

As disclosed herein, N-acetylglucosaminyltransferase II (GlcNAc-TII;GnTII; EC 2.4.1.143) catalyzes the reactionUDP-N-acetyl-D-glucosamine+6-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>UDP+6-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl-R,where R represents the remainder of the N-linked oligosaccharide in theglycan acceptor. An N-acetylglucosaminyltransferase II catalytic domainis any portion of an N-acetylglucosaminyltransferase II enzyme that iscapable of catalyzing this reaction. Amino acid sequences forN-acetylglucosaminyltransferase II enzymes from various organisms arelisted in WO2012069593. In certain embodiments, theN-acetylglucosaminyltransferase II catalytic domain is from the humanN-acetylglucosaminyltransferase II enzyme (SEQ ID NO: 39) or variantsthereof. Additional GnTII species are listed in the CAZy database in theglycosyltransferase family 16 (cazy.org/GT16_all). Enzymaticallycharacterized species include GnTII of C. elegans, D. melanogaster, Homosapiens (NP_002399.1), Rattus norvegicus, Sus scrofa(cazy.org/GT16_characterized). In certain embodiments, theN-acetylglucosaminyltransferase II catalytic domain contains a sequencethat is at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to amino acid residues from about 30 to about 447of SEQ ID NO: 39. The catalytic domain may include N-terminal parts ofthe enzyme such as all or part of the stem domain, the transmembranedomain, or the cytoplasmic domain.

In embodiments where the filamentous fungal cell contains a fusionprotein of the invention, the fusion protein may further contain aspacer in between the N-acetylglucosaminyltransferase I catalytic domainand the N-acetylglucosaminyltransferase II catalytic domain. In certainembodiments, the spacer is an EGIV spacer, a 2×G4S spacer, a 3×G4Sspacer, or a CBHI spacer. In other embodiments, the spacer contains asequence from a stem domain.

For ER/Golgi expression the N-acetylglucosaminyltransferase I and/orN-acetylglucosaminyltransferase II catalytic domain is typically fusedwith a targeting peptide or a part of an ER or early Golgi protein, orexpressed with an endogenous ER targeting structures of an animal orplant N-acetylglucosaminyltransferase enzyme. In certain preferredembodiments, the N-acetylglucosaminyltransferase I and/orN-acetylglucosaminyltransferase II catalytic domain contains any of thetargeting peptides of the invention as described in the section entitled“Targeting sequences”. Preferably, the targeting peptide is linked tothe N-terminal end of the catalytic domain. In some embodiments, thetargeting peptide contains any of the stem domains of the invention asdescribed in the section entitled “Targeting sequences”. In certainpreferred embodiments, the targeting peptide is a Kre2/Mnt1 targetingpeptide. In other embodiments, the targeting peptide further contains atransmembrane domain linked to the N-terminal end of the stem domain ora cytoplasmic domain linked to the N-terminal end of the stem domain. Inembodiments where the targeting peptide further contains a transmembranedomain, the targeting peptide may further contain a cytoplasmic domainlinked to the N-terminal end of the transmembrane domain.

The filamentous fungal cells may also contain a polynucleotide encodinga UDP-GlcNAc transporter. The polynucleotide encoding the UDP-GlcNActransporter may be endogenous (i.e., naturally present) in the hostcell, or it may be heterologous to the filamentous fungal cell.

In certain embodiments, the filamentous fungal cell may further containa polynucleotide encoding a α-1,2-mannosidase. The polynucleotideencoding the α-1,2-mannosidase may be endogenous in the host cell, or itmay be heterologous to the host cell. Heterologous polynucleotides areespecially useful for a host cell expressing high-mannose glycanstransferred from the Golgi to the ER without effectiveexo-α-2-mannosidase cleavage. The α-1,2-mannosidase may be α mannosidaseI type enzyme belonging to the glycoside hydrolase family 47(cazy.org/GH47_all.html). In certain embodiments the α-1,2-mannosidaseis an enzyme listed at cazy.org/GH47_characterized.html. In particular,the α-1,2-mannosidase may be an ER-type enzyme that cleavesglycoproteins such as enzymes in the subfamily of ER α-mannosidase I EC3.2.1.113 enzymes. Examples of such enzymes include humanα-2-mannosidase 1B (AAC26169), a combination of mammalian ERmannosidases, or a filamentous fungal enzyme such as α-1,2-mannosidase(MDS1) (T. reesei AAF34579; Maras M et al J Biotech. 77, 2000, 255, orTrire 45717). For ER expression, the catalytic domain of the mannosidaseis typically fused with a targeting peptide, such as HDEL, KDEL, or partof an ER or early Golgi protein, or expressed with an endogenous ERtargeting structures of an animal or plant mannosidase I enzyme.

In certain embodiments, the filamentous fungal cell may also furthercontain a polynucleotide encoding a galactosyltransferase.Galactosyltransferases transfer β-linked galactosyl residues to terminalN-acetylglucosaminyl residue. In certain embodiments thegalactosyltransferase is a β-1,4-galactosyltransferase. Generally,β-1,4-galactosyltransferases belong to the CAZy glycosyltransferasefamily 7 (cazy.org/GT7_all.html) and includeβ-N-acetylglucosaminyl-glycopeptide β-1,4-galactosyltransferase (EC2.4.1.38), which is also known as N-acetylactosamine synthase (EC2.4.1.90). Useful subfamilies include β4-GalT1, β4-GalT-I, -III, -IV,-V, and -VI, such as mammalian or human β4-GalTI or β4GalT-II, -III,-IV, -V, and -VI or any combinations thereof. β4-GalT1, β4-GalTII, orβ4-GalTIII are especially useful for galactosylation of terminalGlcNAcβ2-structures on N-glycans such as GlcNAcMan3, GlcNAc2Man3, orGlcNAcMan5 (Guo S. et al. Glycobiology 2001, 11:813-20). Thethree-dimensional structure of the catalytic region is known (e.g.(2006) J. Mol. Biol. 357: 1619-1633), and the structure has beenrepresented in the PDB database with code 2FYD. The CAZy databaseincludes examples of certain enzymes. Characterized enzymes are alsolisted in the CAZy database at cazy.org/GT7_characterized.html. Examplesof useful β4GalT enzymes include β4GalT1, e.g. bovine Bos taurus enzymeAAA30534.1 (Shaper N. L. et al Proc. Natl. Acad. Sci. U.S.A. 83 (6),1573-1577 (1986)), human enzyme (Guo S. et al. Glycobiology 2001,11:813-20), and Mus musculus enzyme AAA37297 (Shaper, N. L. et al. 1998J. Biol. Chem. 263 (21), 10420-10428); β4GalTII enzymes such as humanβ4GalTII BAA75819.1, Chinese hamster Cricetulus griseus AAM77195, Musmusculus enzyme BAA34385, and Japanese Medaka fish Oryzias latipesBAH36754; and β4GaTIII enzymes such as human β4GaTIII BAA75820.1,Chinese hamster Cricetulus griseus AAM77196 and Mus musculus enzymeAAF22221.

The galactosyltransferase may be expressed in the plasma membrane of thehost cell. A heterologous targeting peptide, such as a Kre2 peptidedescribed in Schwientek J. Biol. Chem 1996 3398, may be used. Promotersthat may be used for expression of the galactosyltransferase includeconstitutive promoters such as gpd, promoters of endogenousglycosylation enzymes and glycosyltransferases such asmannosyltransferases that synthesize N-glycans in the Golgi or ER, andinducible promoters of high-yield endogenous proteins such as the cbh1promoter.

In certain embodiments of the invention where the filamentous fungalcell contains a polynucleotide encoding a galactosyltransferase, thefilamentous fungal cell also contains a polynucleotide encoding aUDP-Gal 4 epimerase and/or UDP-Gal transporter. In certain embodimentsof the invention where the filamentous fungal cell contains apolynucleotide encoding a galactosyltransferase, lactose may be used asthe carbon source instead of glucose when culturing the host cell. Theculture medium may be between pH 4.5 and 7.0 or between 5.0 and 6.5. Incertain embodiments of the invention where the filamentous fungal cellcontains a polynucleotide encoding a galactosyltransferase and apolynucleotide encoding a UDP-Gal 4 epimerase and/or UDP-Galtransporter, a divalent cation such as Mn2+, Ca2+ or Mg2+ may be addedto the cell culture medium.

Accordingly, in certain embodiments, the filamentous fungal cell of theinvention, for example, selected among Neurospora, Trichoderma,Myceliophthora or Chrysosporium cell, and more preferably Trichodermareesei cell, may comprise the following features:

-   a) a mutation in at least one endogenous protease that reduces or    eliminates the activity of said endogenous protease, preferably the    protease activity of two or three or more endogenous proteases is    reduced, for example, pep1, tsp1, gap1 and/or slp1 proteases, in    order to improve production or stability of a heterologous protein    to be produced,-   b) a mutation in a PMT gene, for example T. reesei pmt1 gene, that    reduces or eliminates endogenous O-mannosyltransferase activity    compared to a parental Trichoderma cell which does not have said    second mutation,-   c) a polynucleotide encoding a protein having at least one serine or    threonine, preferably a heterologous glycoprotein, such as an    immunoglobulin, an antibody, or a protein fusion comprising Fc    fragment of an immunoglobulin.-   d) optionally, a deletion or disruption of the alg3 gene,-   e) optionally, a polynucleotide encoding    N-acetylglucosaminyltransferase I catalytic domain and a    polynucleotide encoding N-acetylglucosaminyltransferase catalytic    domain,-   f) optionally, a polynucleotide encoding β1,4 galactosyltransferase,-   g) optionally, a polynucleotide or polynucleotides encoding UDP-Gal    4 epimerase and/or transporter.

Targeting Sequences

In certain embodiments, recombinant enzymes, such as α1,2 mannosidases,GnTI, or other glycosyltransferases introduced into the filamentousfungal cells, include a targeting peptide linked to the catalyticdomains. The term “linked” as used herein means that two polymers ofamino acid residues in the case of a polypeptide or two polymers ofnucleotides in the case of a polynucleotide are either coupled directlyadjacent to each other or are within the same polypeptide orpolynucleotide but are separated by intervening amino acid residues ornucleotides. A “targeting peptide”, as used herein, refers to any numberof consecutive amino acid residues of the recombinant protein that arecapable of localizing the recombinant protein to the endoplasmicreticulum (ER) or Golgi apparatus (Golgi) within the host cell. Thetargeting peptide may be N-terminal or C-terminal to the catalyticdomains. In certain embodiments, the targeting peptide is N-terminal tothe catalytic domains. In certain embodiments, the targeting peptideprovides binding to an ER or Golgi component, such as to α mannosidaseenzyme. In other embodiments, the targeting peptide provides directbinding to the ER or Golgi membrane.

Components of the targeting peptide may come from any enzyme thatnormally resides in the ER or Golgi apparatus. Such enzymes includemannosidases, mannosyltransferases, glycosyltransferases, Type 2 Golgiproteins, and MNN2, MNN4, MNN6, MNN9, MNN10, MNS1, KRE2, VAN1, and OCH1enzymes. Such enzymes may come from a yeast or fungal species such asthose of Acremonium, Aspergillus, Aureobasidium, Cryptococcus,Chrysosporium, Chrysosporium lucknowense, Filobasidium, Fusarium,Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Myrothecium,Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, andTrichoderma. Sequences for such enzymes can be found in the GenBanksequence database.

In certain embodiments the targeting peptide comes from the same enzymeand organism as one of the catalytic domains of the recombinant protein.For example, if the recombinant protein includes a human GnTII catalyticdomain, the targeting peptide of the recombinant protein is from thehuman GnTII enzyme. In other embodiments, the targeting peptide may comefrom a different enzyme and/or organism as the catalytic domains of therecombinant protein.

Examples of various targeting peptides for use in targeting proteins tothe ER or Golgi that may be used for targeting the recombinant enzymes,include: Kre2/Mnt1 N-terminal peptide fused to galactosyltransferase(Schwientek, J B C 1996, 3398), HDEL for localization of mannosidase toER of yeast cells to produce Man5 (Chiba, J B C 1998, 26298-304;Callewaert, FEBS Lett 2001, 173-178), OCH1 targeting peptide fused toGnTI catalytic domain (Yoshida et al, Glycobiology 1999, 53-8), yeastN-terminal peptide of Mns1 fused to α2-mannosidase (Martinet et al,Biotech Lett 1998, 1171), N-terminal portion of Kre2 linked to catalyticdomain of GnTI or β4GalT (Vervecken, Appl. Environ Microb 2004,2639-46), various approaches reviewed in Wildt and Gerngross (Nature RevBiotech 2005, 119), full-length GnTI in Aspergillus nidulans (Kalsner etal, Glycocon. J 1995, 360-370), full-length GnTI in Aspergillus oryzae(Kasajima et al, Biosci Biotech Biochem 2006, 2662-8), portion of yeastSec12 localization structure fused to C. elegans GnTI in Aspergillus(Kainz et al 2008), N-terminal portion of yeast Mnn9 fused to human GnTIin Aspergillus (Kainz et al 2008), N-terminal portion of AspergillusMnn10 fused to human GnTI (Kainz et al, Appl. Environ Microb 2008,1076-86), and full-length human GnTI in T. reesei (Maras et al, FEBSLett 1999, 365-70).

In certain embodiments the targeting peptide is an N-terminal portion ofthe Mnt1/Kre2 targeting peptide having the amino acid sequence of SEQ IDNO: 40 (for example encoded by the polynucleotide of SEQ ID NO:41). Incertain embodiments, the targeting peptide is selected from human GNT2,KRE2, KRE2-like, Och1, Anp1, Van1 as shown in the Table 1 below:

TABLE 1 Amino acid sequence of targeting peptides Protein TreIDAmino acid sequence human — MRFRIYKRKVLILTLVVAACGFVLWSSNG GNT2RQRKNEALAPPLLDAEPARGAGGRGGDHP (SEQ ID NO: 42) KRE2 21576MASTNARYVRYLLIAFFTILVFYFVSNSK YEGVDLNKGTFTAPDSTKTTPK (SEQ ID NO: 43)KRE2- 69211 MAIARPVRALGGLAAILWCFFLYQLLRPS like SSYNSPGDRYINFERDPNLDPTG(SEQ ID NO: 44) Och1 65646 MLNPRRALIAAAFILTVFFLISRSHNSESASTS (SEQ ID NO: 45) Anp1 82551 MMPRHHSSGFSNGYPRADTFEISPHRFQPRATLPPHRKRKRTAIRVGIAVVVILVLVL WFGQPRSVASLISLGILSGYDDLKLE (SEQ ID NO: 46)Van1 81211 MLLPKGGLDWRSARAQIPPTRALWNAVTR TRFILLVGITGLILLLWRGVSTSASE(SEQ ID NO: 47)

Further examples of sequences that may be used for targeting peptidesinclude the targeting sequences as described in WO2012/069593.

Uncharacterized sequences may be tested for use as targeting peptides byexpressing enzymes of the glycosylation pathway in a host cell, whereone of the enzymes contains the uncharacterized sequence as the soletargeting peptide, and measuring the glycans produced in view of thecytoplasmic localization of glycan biosynthesis (e.g. as in SchwientekJBC 1996 3398), or by expressing a fluorescent reporter protein fusedwith the targeting peptide, and analysing the localization of theprotein in the Golgi by immunofluorescence or by fractionating thecytoplasmic membranes of the Golgi and measuring the location of theprotein.

Methods for Producing a Protein Having Reduced O-Mannosylation

The filamentous fungal cells as described above are useful in methodsfor producing a protein having reduced O-mannosylation.

Accordingly, in another aspect, the invention relates to a method forproducing a protein having reduced O-mannosylation, comprising:

-   -   a) providing a PMT-deficient Trichoderma cell having a mutation        in a PMT gene that reduces endogenous protein        O-mannosyltransferase activity as compared to parental strain        which does not have such mutation, and further comprising a        polynucleotide encoding a protein with serine or threonine,        which may be O-mannosylated,    -   b) culturing said PMT-deficient Trichoderma cell to produce said        protein having reduced O-mannosylation.

In such method, the produced protein has reduced O-mannosylation due tosaid mutation in said PMT gene as described in the previous sections.The PMT-deficient Trichoderma cell may optionally have reducedendogenous protease activity as described in the previous sections.

The filamentous fungal cells and methods of the invention are useful forthe production of protein with serine or threonine which may beO-mannosylated. For example, it is particularly useful for theproduction of protein which are O-mannosylated when produced in aparental PMT-functional filamentous fungal host cell, for example, in atleast one Trichoderma cell which is wild type for PMT1 gene, such as SEQID NO:1.

In methods of the invention, certain growth media include, for example,common commercially-prepared media such as Luria-Bertani (LB) broth,Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth. Other definedor synthetic growth media may also be used and the appropriate mediumfor growth of the particular host cell will be known by someone skilledin the art of microbiology or fermentation science. Culture mediumtypically has the Trichoderma reesei minimal medium (Penttilä et al.,1987, Gene 61, 155-164) as a basis, supplemented with substancesinducing the production promoter such as lactose, cellulose, spent grainor sophorose. Temperature ranges and other conditions suitable forgrowth are known in the art (see, e.g., Bailey and Ollis 1986). Incertain embodiments the pH of cell culture is between 3.5 and 7.5,between 4.0 and 7.0, between 4.5 and 6.5, between 5 and 5.5, or at 5.5.In certain embodiments, to produce an antibody the filamentous fungalcell or Trichoderma fungal cell is cultured at a pH range selected from4.7 to 6.5; pH 4.8 to 6.0; pH 4.9 to 5.9; and pH 5.0 to 5.8.

In some embodiments, the protein which may be O-mannosylated is aheterologous protein, preferably a mammalian protein. In otherembodiments, the heterologous protein is a non-mammalian protein.

In certain embodiments, the protein which may be O-mannosylated is aglycoprotein with N-glycan posttranslational modifications.

In certain embodiments, a mammalian protein which may be O-mannosylatedis selected from an immunoglobulin, immunoglobulin or antibody heavy orlight chain, a monoclonal antibody, a Fab fragment, an F(ab′)2 antibodyfragment, a single chain antibody, a monomeric or multimeric singledomain antibody, a camelid antibody, or their antigen-binding fragments.

A fragment of a protein, as used herein, consists of at least 10, 20,30, 40, 50, 60, 70, 80, 90, 100 consecutive amino acids of a referenceprotein.

As used herein, an “immunoglobulin” refers to a multimeric proteincontaining a heavy chain and a light chain covalently coupled togetherand capable of specifically combining with antigen. Immunoglobulinmolecules are a large family of molecules that include several types ofmolecules such as IgM, IgD, IgG, IgA, and IgE.

As used herein, an “antibody” refers to intact immunoglobulin molecules,as well as fragments thereof which are capable of binding an antigen.These include hybrid (chimeric) antibody molecules (see, e.g., Winter etal. Nature 349:293-99225, 1991; and U.S. Pat. No. 4,816,567 226);F(ab′)2 molecules; non-covalent heterodimers; dimeric and trimericantibody fragment constructs; humanized antibody molecules (see e.g.,Riechmann et al. Nature 332, 323-27, 1988; Verhoeyan et al. Science 239,1534-36, 1988; and GB 2,276,169); and any functional fragments obtainedfrom such molecules, as well as antibodies obtained throughnon-conventional processes such as phage display or transgenic mice.Preferably, the antibodies are classical antibodies with Fc region.Methods of manufacturing antibodies are well known in the art.

In further embodiments, the yield of the mammalian glycoprotein is atleast 0.5, at least 1, at least 2, at least 3, at least 4, or at least 5grams per liter.

In certain embodiments, the mammalian glycoprotein is an antibody,optionally, IgG1, IgG2, IgG3, or IgG4. In further embodiments, the yieldof the antibody is at least 0.5, at least 1, at least 2, at least 3, atleast 4, or at least 5 grams per liter. In further embodiments, themammalian glycoprotein is an antibody, and the antibody contains atleast 70%, at least 80%, at least 90%, at least 95%, or at least 98% ofa natural antibody C-terminus and N-terminus without additional aminoacid residues. In other embodiments, the mammalian glycoprotein is anantibody, and the antibody contains at least 70%, at least 80%, at least90%, at least 95%, or at least 98% of a natural antibody C-terminus andN-terminus that do not lack any C-terminal or N-terminal amino acidresidues.

In certain embodiments where the mammalian glycoprotein is purified fromcell culture, the culture containing the mammalian glycoprotein containspolypeptide fragments that make up a mass percentage that is less than50%, less than 40%, less than 30%, less than 20%, or less than 10% ofthe mass of the produced polypeptides. In certain preferred embodiments,the mammalian glycoprotein is an antibody, and the polypeptide fragmentsare heavy chain fragments and/or light chain fragments. In otherembodiments, where the mammalian glycoprotein is an antibody and theantibody purified from cell culture, the culture containing the antibodycontains free heavy chains and/or free light chains that make up a masspercentage that is less than 50%, less than 40%, less than 30%, lessthan 20%, or less than 10% of the mass of the produced antibody. Methodsof determining the mass percentage of polypeptide fragments are wellknown in the art and include, measuring signal intensity from anSDS-gel.

In certain embodiments, where the protein with reduced O-mannosylation,e.g. an antibody, is purified from cell culture, the culture contains atleast 70%, 80%, 90%, 95% or 100% of the proteins that is notO-mannosylated (mol %, as determined for example by MALDI TOF MSanalysis, and measuring area or intensity of peaks as described in theExample 1 below).

In certain embodiments where the protein with at least one serine orthreonine residue which may be O-mannosylated is purified from cellculture, and where the strain is a Trichoderma cell geneticallyengineered to produce complex N-glycans, the culture further comprisesat least 5%, 10%, 15%, 20%, 25%, 30% of secreted complex neutralN-glycans (mol %) compared to total secreted neutral N-glycans (asmeasured for example as described in WO2012069593).

In other embodiments, the heterologous protein with reducedO-mannosylation, for example, the antibody, comprises the trimannosylN-glycan structure Manα3[Manα6]Manβ4GlcNAcβ4GlcNAc. In some embodiments,the Manα3[Manα6]Manβ4GlcNAcβ4GlcNAc structure represents at least 20%,30%; 40%, 50%; 60%, 70%, 80% (mol %) or more, of the total N-glycans ofthe heterologous protein with reduced O-mannosylation. In otherembodiments, the heterologous protein with reduced O-mannosylationcomprises the G0 N-glycan structureGlcNAcβ2Manα3[GlcNAcβ2Manα6]Manβ4GlcNAcβ4GlcNAc. In other embodiments,the non-fucosylated G0 glycoform structure represents at least 20%, 30%;40%, 50%; 60%, 70%, 80% (mol %) or more, of the total N-glycans of theheterologous protein with reduced O-mannosylation. In other embodiments,galactosylated N-glycans represents less (mol %) than 0.5%, 0.1%, 0.05%,0.01% of total N-glycans of the culture, and/or of the heterologousprotein with reduced O-mannosylation, for example an antibody. Incertain embodiments, the culture or the heterologous protein, forexample an antibody, comprises no galactosylated N-glycans.

In certain embodiments, the heterologous (purified) protein is anantibody, a light chain antibody, a heavy chain antibody or a Fab, thatcomprises Man3, GlcNAcMan3, Man5, GlcNAcMan5, G0, core G0, G1, or G2N-glycan structure as major glycoform and less than 35%, 20%, 17%, 15%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, or less than0.5% of O-mannosylation level (as mole % as determined for example byMALDI TOF MS analysis, and measuring area or intensity of peaks asdescribed in Example 1).

In a specific embodiment, the invention therefore relates to a methodfor producing an antibody having reduced O-mannosylation, comprising:

-   -   a. providing a PMT-deficient Trichoderma cell having        -   i. a mutation that reduces endogenous protein            O-mannosyltransferase activity as compared to parental            strain which does not have such mutation and        -   ii. a polynucleotide encoding a light chain antibody and a            polynucleotide encoding a heavy chain antibody,    -   b. culturing the cell to produce said antibody, consisting of        heavy and light chains, having reduced O-mannosylation.

In such specific embodiments of the methods related to the production ofantibody, at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% of theproduced antibody is not O-mannosylated (mol %, as determined forexample by MALDI TOF MS analysis, and measuring area or intensity ofpeaks as described in Example 1.

In certain embodiments of any of the disclosed methods, the methodincludes the further step of providing one or more, two or more, threeor more, four or more, or five or more protease inhibitors. In certainembodiments, the protease inhibitors are peptides that are co-expressedwith the mammalian polypeptide. In other embodiments, the inhibitorsinhibit at least two, at least three, or at least four proteases from aprotease family selected from aspartic proteases, trypsin-like serineproteases, subtilisin proteases, and glutamic proteases.

In certain embodiments of any of the disclosed methods, the filamentousfungal cell or Trichoderma fungal cell also contains a carrier protein.As used herein, a “carrier protein” is portion of a protein that isendogenous to and highly secreted by a filamentous fungal cell orTrichoderma fungal cell. Suitable carrier proteins include, withoutlimitation, those of T. reesei mannanase I (Man5A, or MANI), T. reeseicellobiohydrolase II (Cel6A, or CBHII) (see, e.g., Paloheimo et al Appl.Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T. reeseicellobiohydrolase I (CBHI). In some embodiments, the carrier protein isCBH1. In other embodiments, the carrier protein is a truncated T. reeseiCBH1 protein that includes the CBH1 core region and part of the CBH1linker region. In some embodiments, a carrier such as acellobiohydrolase or its fragment is fused to an antibody light chainand/or an antibody heavy chain. In some embodiments, a carrier-antibodyfusion polypeptide comprises a Kex2 cleavage site. In certainembodiments, Kex2, or other carrier cleaving enzyme, is endogenous to afilamentous fungal cell. In certain embodiments, carrier cleavingprotease is heterologous to the filamentous fungal cell, for example,another Kex2 protein derived from yeast or a TEV protease. In certainembodiments, carrier cleaving enzyme is overexpressed. In certainembodiments, the carrier consists of about 469 to 478 amino acids ofN-terminal part of the T. reesei CBH1 protein GenBank accession No.EGR44817.1.

In certain embodiments, the filamentous fungal cell of the inventionoverexpress KEX2 protease. In an embodiment the heterologous protein isexpressed as fusion construct comprising an endogenous fungalpolypeptide, a protease site such as a Kex2 cleavage site, and theheterologous protein such as an antibody heavy and/or light chain.Useful 2-7 amino acids combinations preceding Kex2 cleavage site havebeen described, for example, in Mikosch et al. (1996) J. Biotechnol.52:97-106; Goller et al. (1998) Appl Environ Microbiol. 64:3202-3208;Spencer et al. (1998) Eur. J. Biochem. 258:107-112; Jalving et al.(2000) Appl. Environ. Microbiol. 66:363-368; Ward et al. (2004) Appl.Environ. Microbiol. 70:2567-2576; Ahn et al. (2004) Appl. Microbiol.Biotechnol. 64:833-839; Paloheimo et al. (2007) Appl Environ Microbiol.73:3215-3224; Paloheimo et al. (2003) Appl Environ Microbiol.69:7073-7082; and Margolles-Clark et al. (1996) Eur J Biochem.237:553-560.

The invention further relates to the protein composition, for examplethe antibody composition, obtainable or obtained by the method asdisclosed above.

In specific embodiment, such antibody composition obtainable or obtainedby the methods of the invention, comprises at least 70%, 80%, 90%, 95%,or 100% of the antibodies that are not O-mannosylated (mol %, asdetermined for example by MALDI TOF MS analysis, and measuring area orintensity of peaks as described in Example 1). In other specificembodiments, such antibody composition further comprises as 50%, 60%,70% or 80% (mole % neutral N-glycan), of the following glycoform:

-   -   (i) Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc (Man5 glycoform);    -   (ii) GlcNAcβ2Manα3[Manα6(Manα3)Manα6]Manβ4GlcNAβ4GlcNAc, or        β4-galactosylated variant thereof;    -   (iii) Manα6(Manα3)Manβ4GlcNAβ4GlcNAc;    -   (iv) Manα6(GlcNAcβ2Manα3)Manβ4GlcNAβ4GlcNAc, or        β4-galactosylated variant thereof: or,    -   (v) complex type N-glycans selected from the G0, G1 or G2        glycoform.

In some embodiments the N-glycan glycoform according to iii-v comprisesless than 15%, 10%, 7%, 5%, 3%, 1% or 0.5% or is devoid of Man5 glycanas defined in i) above.

The invention also relates to a method of reducing O-mannosylation levelof a recombinant glycoprotein composition produced in a Trichodermacell, said method consisting of using a Trichoderma cell having amutation in a PMT gene wherein said PMT gene is either:

-   -   a. PMT1 gene comprising the polynucleotide of SEQ ID NO:1,    -   b. a functional homologous gene of PMT1 gene, which gene is        capable of restoring parental O-mannosylation level by        functional complementation when introduced into a T. reesei        strain having a disruption in said PMT1 gene, or,    -   c. a polynucleotide encoding a polypeptide having at least 50%,        at least 60%, at least 70%, at least 90%, or at least 95%        identity with SEQ ID NO:2, said polypeptide having protein        O-mannosyltransferase activity.

In one specific embodiment of such method, said Trichoderma cell isTrichoderma reesei.

In another specific embodiment of such method, said recombinantglycoprotein comprises at least a light chain antibody or its fragmentscomprising at least one serine or threonine residue and with at leastone N-glycan.

EXAMPLES

As more specifically exemplified in Example 2, after deletion of pmt1,almost 95% of purified mAb and 70% of Fab molecules no longer containedany O-mannose residues. In contrast, as exemplified in Examples 3 to 4,O-mannosylation level analysis performed on pmt2 and pmt3 deletionstrains did not exhibit any appreciable reduction in O-mannosylation.Together with the titer and growth analysis set forth in Example 2,these results demonstrate that filamentous fungal cells, such asTrichoderma cells, can be genetically modified to reduce or suppressO-mannosylation activity, without adversely affecting viability andyield of produced glycoproteins. As such, pmt1 is identified a valuabletarget to reduce O-mannosylation of secreted proteins and to improveproduct quality of biopharmaceuticals produced by Trichoderma reesei.

Example 1: Pmt1 Deletion in a Trichoderma reesei Strain

This example demonstrates that pmt1 is a valuable target to reduceO-mannosylation of secreted proteins and to improve product quality ofbiopharmaceuticals produced by Trichoderma reesei.

Generation of Pmt1 Deletion Plasmids

Three different deletion plasmids (pTTv36, pTTv124, pTTv185) wereconstructed for deletion of the protein O-mannosyltransferase gene pmt1(TreD75421). All the plasmids contain the same 5′ and 3′ flankingregions for correct integration to the pmt1 locus. The differencebetween the three plasmids is the marker used in the selection; pTTv36contains a gene encoding acetamidase of Aspergillus nidulans (amdS),pTTv124 contains a loopout version (blaster cassette) of the amdS markerand pTTv185 a loopout version (blaster cassette) of a gene encodingorotidine-5′-monophosphate (OMP) decarboxylase of T. reesei(pyr4).

The third deletion construct, pTTv185, for the proteinO-mannosyltransferase gene pmt1 (TreID75421) was designed to enableremoval of the selection marker from the Trichoderma reesei genome aftersuccessful integration and thereby recycling of the selection marker forsubsequent transformations. In this approach, the recycling of themarker, i.e. removal of pyr4 gene from the deletion construct, resemblesso called blaster cassettes developed for yeasts (Hartl, L. and Seiboth,B., 2005, Curr Genet 48:204-211; and Alani, E. et al., 1987, Genetics116:541-545). Similar blaster cassettes have also been developed forfilamentous fungi including Hypocrea jecorina (anamorph: T. reesei)(Hartl, L. and Seiboth, B., 2005, Curr Genet 48:204-211).

The TreID number refers to the identification number of a particularprotease gene from the Joint Genome Institute Trichoderma reesei v2.0genome database. Primers for construction of deletion plasmids weredesigned either manually or using Primer3 software (Primer3 website,Rozen and Skaletsky (2000) Bioinformatics Methods and Protocols: Methodsin Molecular Biology. Humana Press, Totowa, N.J., pp 365-386).

The principle of the blaster cassette using pyr4 as the marker gene isas follows: pyr4, encoding orotidine-5′-monophosphate (OMP)decarboxylase of T. reesei (Smith, J. L., et al., 1991, Current Genetics19:27-33) is needed for uridine synthesis. Strains deficient for OMPdecarboxylase activity are unable to grow on minimal medium withouturidine supplementation (i.e. are uridine auxotrophs). The utilisationof 5-fluoroorotic acid (5-FOA) in generation of mutant strains lackingOMP decarboxylase activity (pyr4⁻ strains) is based on the conversion of5-FOA to a toxic intermediate 5-fluoro-UMP by OMP decarboxylase.Therefore, cells which have a mutated pyr4 gene are resistant to 5-FOA,but in addition are also auxotrophic for uridine. The 5-FOA resistancecan in principle result also from a mutation in another gene (pyr2,orotate phosphoribosyltransferase), and therefore the spontaneousmutants obtained with this selection need to be verified for thepyr4-genotype by complementing the mutant with the pyr4 gene. Oncemutated, the pyr4 gene can be used as an auxotrophic selection marker inT. reesei. In our blaster cassette pyr4 is followed by a 310 bp directrepeat of pyr4 5′ untranslated region (5′UTR) and surrounded by 5′ and3′ flanking regions of the gene to be deleted. Integration of thedeletion cassette is selected via the pyr4 function. Removal of the pyr4marker is then forced in the presence of 5-FOA by recombination betweenthe two homologous regions (direct repeat of 5′UTR) resulting in loopingout of the selection marker and enabling the utilisation of the sameblaster cassette (pyr4 loopout) in successive rounds of gene deletions.After looping out, only the 310 bp sequence of 5′UTR remains in thelocus.

Thus, the pyr4 selection marker and the 5′ direct repeat (DR) fragment(310 bp of pyr4 5′UTR) were produced by PCR using plasmid containing agenomic copy of T. reesei pyr4 as a template. Both fragments contained40 bp overlapping sequences needed to clone the plasmid with the loopoutcassette using homologous recombination in yeast (see below). To enablepossible additional cloning steps, an AscI digestion site was placedbetween the pyr4 marker and the 5′ direct repeat and NotI sites tosurround the complete blaster cassette.

1100 bp of 5′ and 1000 bp of 3′ flanking regions were selected as thebasis of the pmt1 deletion plasmids. The flanking region fragments wereproduced by PCR using a T. reesei wild type strain QM6a (ATCC13631) asthe template. For the yeast homologous recombination system used incloning (see below), overlapping sequences for the vector and theselection marker were placed to the appropriate PCR-primers. To enablemarker switch in the construct, NotI restriction sites were introducedbetween the flanking regions and the selection marker. PmeI restrictionsites were placed between the vector and the flanking regions forremoval of vector sequence prior to transformation into T. reesei.Vector backbone pRS426 was digested with restriction enzymes (EcoRI andXhoI).

First deletion plasmid for pmt1 (plasmid pTTv36, Table 2) used amdS, agene encoding acetamidase of Aspergillus nidulans, as the marker. Themarker cassette was digested from an existing plasmid pHHO1 with NotI.All fragments used in cloning were separated with agarose gelelectrophoresis and correct fragments were isolated from the gel with agel extraction kit (Qiagen) using standard laboratory methods.

To construct the first deletion plasmid pTTv36, the vector backbone andthe appropriate marker and flanking region fragments were transformedinto Saccharomyces cerevisiae (strain H3488/FY834). The yeasttransformation protocol was based on the method for homologous yeastrecombination described in the Neurospora knockouts workshop material ofColot and Collopy, (Dartmouth Neurospora genome protocols website), andthe Gietz laboratory protocol (University of Manitoba, Gietz laboratorywebsite). The plasmid DNA from the yeast transformants was rescued bytransformation into Escherichia coli. A few clones were cultivated,plasmid DNA was isolated and digested to screen for correctrecombination using standard laboratory methods. A few clones withcorrect insert sizes were sequenced and stored.

To clone the second pmt1 deletion plasmid (pTTv124, Table 2), the amdSmarker was removed from the deletion plasmid pTTv36 with NotI digestionand replaced by another variant of the blaster cassette, amdS loopoutcassette containing the amdS selection marker gene, followed by AscIrestriction site and a 300 bp direct repeat of amdS 5′UTR. The amdSblaster cassette functions in a similar manner to the pyr4 blastercassette. The clones containing the amdS blaster cassette are able togrow on acetamide as sole nitrogen source. On medium containing5-fluoroacetamide (5-FAA) a functional amdS gene will convert 5-FAA to atoxic fluoroacetate and therefore, in the presence of 5-FAA, removal ofamdS gene is beneficial to the fungus. Removal of amdS blaster cassetteis enhanced via the 300 bp DRs in the cassette like in the pyr4 blastercassette, which enables the amdS gene to loop out via single crossoverbetween the two DRs. Resulting clones are resistant to 5-FAA and unableto grow on acetamide as the sole nitrogen source.

The fragments needed for the amdS blaster cassette were produced by PCRusing a plasmid β3SR2 (Hynes M. J. et al, 1983, Mol. Cell. Biol.3:1430-1439) containing a genomic copy of the amdS gene as the template.For the yeast homologous recombination system used in cloning (seeabove), overlapping sequences were placed to the appropriatePCR-primers. To enable marker switch in the construct, NotI restrictionsites were kept between the flanking regions and the blaster cassette.Additional restriction sites FseI and AsiSI were introduced to the 5′end of amdS and an AscI site between amdS and amdS 5′DR. The plasmidpTTv124 was constructed using the yeast recombination system describedabove. The plasmid DNA from the yeast transformants was rescued bytransformation into Escherichia coli. A few clones were cultivated,plasmid DNA was isolated and digested to screen for correctrecombination using standard laboratory methods. A few clones withcorrect insert sizes were sequenced and stored.

To clone the third pmt1 deletion plasmid (pTTv185, Table 2), the amdSmarker was removed from the deletion plasmid pTTv36 with NotI digestionand replaced by the pyr4 blaster cassette described above. The pyr4blaster cassette was obtained from another plasmid with NotI digestion,ligated to NotI cut pTTv36 and transformed into E. coli using standardlaboratory methods. A few transformants were cultivated, plasmid DNAisolated and digested to screen for correct ligation and orientation ofthe pyr4 blaster cassette using standard laboratory methods. One clonewith correct insert size and orientation was sequenced and stored.

These deletion plasmids for pmt1 (pTTv36, pTTv124 and pTTv185) result in2465 bp deletion in the pmt1 locus and cover the complete codingsequence of PMT1.

TABLE 2 Primers for generating deletion plasmidspTTv36, pTTv124 and pTTv185 for proteinO-mannosyltransferase 1 (pmt1, TreID75421) Primer SequenceDeletion plasmid pTTv36 for pmt1 (TreID75421), vector backbone pRS42675421_5′F CGATTAAGTTGGGTAACGCCAGGGT TTTCCCAGTCACGACGGTTTAAACGCTGCAGGGCGTACAGAACT  (SEQ ID NO: 48) 75421_5′R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGTGCGGCCGCG GCTCTAAAATGCTTCACAG  (SEQ ID NO: 49) 75421_3′FCGGTTCTCATCTGGGCTTGCTCGGT CCTGGCGTAGATCTAGCGGCCGCAC GATGATGATGACAGCCAG (SEQ ID NO: 50) 75421_3′R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGTTTAAACC GTCCAGCTCCCGCAGCGCC  (SEQ ID NO: 51)Deletion plasmid pTTv124 for pmt1 (TreID75421), vector backbone pTTv36T282_75421_amds_5for ATCGCTAACTGCTTTCTCTTCTGTG AAGCATTTTAGAGCCGCGGCCGCGGCCGGCCGCGATCGCCTAGATCTACG CCAGGACCG (SEQ ID NO: 52) T283_amds_3rev_loopCGGTCCTGGCGTAGATCTAGGGCGC GCCACTGGAAACGCAACCCTGAA  (SEQ ID NO: 53)T284_amds_loop_5for TTCAGGGTTGCGTTTCCAGTGGCGC GCCCTAGATCTACGCCAGGACCG (SEQ ID NO: 54) T287_75421_loop_3rev AGCATCATGACCGCCCCCTTCTGGCTGTCATCATCATCGTGCGGCCGCGA TTATTGCACAAGCAGCGA  (SEQ ID NO: 55)Deletion plasmid pTTv185 for pmt1 (TreID75421), vector backbone pTTv36no new primers, pTTv36 digested with NotI andligated with pyr4-loopout fragment obtained from another plasmid

Generation of Pmt1 Deletion Strains M403, M404, M406 and M407

To generate a pyr4 negative target strain suitable for the deletion ofpmt1 using plasmid pTTv185, the MAB01 antibody producing strain M304 wassubjected to selection in the presence of 5-fluoro-orotic acid in orderto select for strains containing impaired pyr4 genes. The generation ofthe strain M304 is described in the International Patent Application No.PCT/EP2013/05012. T. reesei strain M304 comprises MAB01 light chainfused to T. reesei truncated CBH1 carrier with NVISKR Kex2 cleavagesequence, MAB01 heavy chain fused to T. reesei truncated CBH1 carrierwith AXE1 [DGETVVKR] Kex2 cleavage sequence, Δpep1Δtsp1Δslp1, andoverexpresses T. reesei KEX2.

Spores of M304 were spread onto minimal medium plates containing 20 g/lglucose, 2 g/l proteose peptone, 5 mM uridine and 1.5 g/l 5-FOA, pH 4.8.Some 5-FOA resistant colonies were streaked after 5-7 days onto platesdescribed above with 1 ml/l Triton X-100 supplementation. A few cloneswere further purified to single cell clones via consecutive purificationplatings: a small piece of mycelia was picked to 0.8% NaCl-0.025% Tween20-20% glycerol, suspended thoroughly by vortexing and filtrated througha cotton-filled pipette tip. Purified clones were sporulated on platescontaining 39 g/l potato dextrose agarose. These clones were tested foruridine auxotrophy by plating spores onto minimal medium plates (20 g/lglucose, 1 ml/l Triton X-100, pH 4.8) with and without 5 mM uridinesupplementation. No growth was observed on plates without uridineindicating the selected clones were putative pyr4⁻. Clones were storedfor future use and one of them was designated with strain number M317.

Pmt1 was deleted from M317 (pyr4⁻ of the strain M304) using the pmt1deletion cassette from plasmid pTTv185 described above. To remove thevector sequence, plasmid pTTv185 (Δpmt1-pyr4) was digested withPmeI+XbaI and the correct fragment was purified from an agarose gelusing QAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of thepmt1 deletion cassette was used to transform strain M317. Preparation ofprotoplasts and transformation for pyr4 selection were carried outessentially according to methods in Penttilä et al. (1987, Gene61:155-164) and Gruber et al (1990, Curr. Genet. 18:71-76).

100 colonies were picked as selective streaks. 40 transformants werescreened by PCR using the primers in Table 3 for the correct integrationof the deletion cassette using standard laboratory methods. 12 putativedeletion clones were purified to single cell clones. Purified cloneswere rescreened for integration and for deletion of pmt1 ORF usingprimers on Table 5. Four clones (in duplicate) were pure disruptants(i.e. no signal with ORF primers).

TABLE 3 Primers for screening integration ofdeletion cassette pTTv185 and fordeletion of protein O-mannosyltransferase 1(pmt1, TreID75421) from M317. Primer Sequence T296_75421_5intTATGGCTTTAGATGGGGACA (SEQ ID NO: 56) T027_Pyr4_orf_start_revTGCGTCGCCGTCTCGCTCCT (SEQ ID NO: 57) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 58) T297_75421_3int CCTGTATCGTCCTGTTCC(SEQ ID NO: 59) T359_pmt1_orf_for GCGCCTGTCGAGTCGGCATT (SEQ ID NO: 60)T360_pmt1_orf_rev CACCGGCCATGCTCTTGCCA (SEQ ID NO: 61)T756_pmt1_orf_for2 CAAGGTGCCCTATGTCGC (SEQ ID NO: 62) T757_pmt1_orf_rev2GATCGGGTCAGGACGGAA (SEQ ID NO: 63)

Deletion of pmt1 was verified by Southern analyses. DNA for Southernanalyses was extracted with Easy-DNA kit for genomic DNA isolation(Invitrogen) essentially according to the manufacturer's instructions.

Southern analyses were essentially performed according to the protocolfor homologous hybridizations in Sambrook et al. (1989, MolecularCloning: A laboratory manual. 2^(nd) Ed., Cold Spring Harbor LaboratoryPress) using radioactive labeling (³²P-dCTP) and DecaLabel Plus kit(Fermentas). Southern digestion schemes were designed using Geneious Prosoftware (Geneious website). Fragments for probes were produced by PCRusing the primers listed in Table 4 using a T. reesei wild type strainQM6a (ATCC13631) as the template. PCR products were separated withagarose gel electrophoresis and correct fragments were isolated from thegel with a gel extraction kit (Qiagen) using standard laboratorymethods.

TABLE 4 Primers for production of probefragments used in Southern analyses of protein O-mannosyltransferase 1(pmt1, TreID75421) deletion strains. Primer Sequence T635_pmt1_5f_forAGCCTGTCTGAGGGACGG (SEQ ID NO: 64) T636_pmt1_5f_rev CAAGGTCGAGATTCGGCA(SEQ ID NO: 65) T637_pmt1_3f_for CAGAAGGGGGCGGTCAT (SEQ ID NO: 66)T638_pmt1_3f_rev GTCCCAGCTCCCGCTCT (SEQ ID NO: 67) T359_pmt1_orf_forGCGCCTGTCGAGTCGGCATT (SEQ ID NO: 68) T360_pmt1_orf_revCACCGGCCATGCTCTTGCCA (SEQ ID NO: 69)

None of the clones hybridised with pmt1 ORF probe (FIG. 1A) indicatingsuccessful deletion of pmt1. Analyses using 5′ and 3′ flank probesrevealed that four of the clones were single integrants (FIGS. 1B and1C; 26-8A and B, 26-21A and B). Four clones gave additional signals andthus indicated multiple integration of the deletion cassette. Four pureclones (with and without additional copies of the deletion cassette)have been stored for future use (M403; 26-8A, M404; 26-19A, M406; 26-16Band M407; 26-198).

Example 2 Analyses of ΔPmt1 Strains M403, M404, M406 and M407

Shake flask cultivation of T. reesei M304 and eight pmt1 deletionstrains (26-8A (M403), 26-8B, 26-16A, 26-16B (M406), 26-19A (M404),26-19B (M407), 26-21A, 26-21B) was carried out in Trichoderma minimalmedium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, 9g/l casamino acids, pH 5.5 at +28° C., 200 rpm. Samples were collectedon days 3, 5, 7 and 10 by vacuum filtration. Supernatant samples werestored to −20° C. (antibody and glycan analyses) or used in pHdeterminations. Mycelia for cell dry weight determinations were rinsedonce with DDIW and dried at +100° C. for 20-24 h. Mycelia for genomicDNA extraction were rinsed once with DDIW and stored to −20° C.

O-mannosylation status analysis was performed to shake flaskcultivations of T. reesei M304, eight pmt1 disruptants (pTTv185: 26-8A,26-8B, 26-16A, 26-16B, 26-19A, 26-19B, 26-21A, 26-21B). All werecultivated in TrMM—40 g/l lactose—20 g/l SGE—100 mM PIPPS—9 g/l casaminoacids, pH 5.5 at +28° C. and samples were taken on time point days 3, 5,7 and 10.

MAB01 antibody from each sample from day 7 was purified fromsupernatants using Protein G HP MultiTrap 96-well plate (GE Healthcare)according to manufacturer's instructions. The antibody was eluted with0.1 M citrate buffer, pH 2.6 and neutralized with 2 M Tris, pH 9. Theconcentration was determined via UV absorbance in spectrophotometeragainst MAB01 standard curve. For O-mannosylation analysis, 10 μg ofprotein was incubated in 6 M Guanidinium HCl for 30 minutes at +60° C.after which 5 μl of fresh 0.1 M DTT was added and incubated again asabove. The samples were purified using Poros R1 96-well plate and theresulting light chains were analysed using MALDI-TOF MS. All were madeas duplicates.

In flask cultures the O-mannosylation status in pmt1 disruptants wasremarkably changed; all Δpmt1 disruptants looked the same—nearlycomplete loss of O-mannosylation in MAB01 LC (FIG. 2: Spectra of lightchain of flask cultured parental T. reesei strain M317 (pyr4⁻ of M304)(A) and Δpmt1 disruptant clone 26-8A (B), day 7).

Fermentation of ΔPmt1 Strain M403

Fermentation was carried out with Δpmt1 strain M403 (clone 26-8A;pTTv185 in M317). Fermentation culture medium contained 30 g/l glucose,60 g/l lactose, 60 g/l whole spent grain at pH 5.5. Lactose feed wasstarted after glucose exhaustion. Growth temperature was shifted from+28° C. to +22° C. after glucose exhaustion. Samples were collected byvacuum filtration. Supernatant samples were stored to −20° C.

In FIG. 3 is shown the Western analyses of supernatant samples. MAB01heavy and light chains were detected from supernatant after day three.Despite the deletion of pmt1, that could also reduce O-mannosylation ofthe linker and thus aid KEX2 cleavage, substantial amount of light chainremains attached to the carrier in the early days of the fermentation.At later stages, the cleavage is more complete but the yield may beaffected by the degradation of the heavy chain. Results on antibodytitres (Table 7 below) indicate fairly steady expression between days 7to 10. In this fermentation the pmt1 deletion strain producedapproximately equal antibody levels as the parental strain. Highertitres were obtained when the same strain was fermented using adifferent fermenter.

M403 (clone 26-8A) was cultivated in fermenter in TrMM, 30 g/l glucose,60 g/l lactose, 60 g/l spent grain, pH 5.5 with lactose feed. Sampleswere harvested on days 2, 3 and 5-11. O-mannosylation level analysis wasperformed as to flask cultures. The O-mannosylation status was greatlydecreased also in fermenter culture (FIG. 4, Table 5).

The O-mannosylation level was calculated from average of area andintensity (Table 5). Area (Table 6) seems to give more commonly higherrate of non-O-glycosylated LC than intensity (Table 7). In all timepoints the O-mannosylation level was below 5%.

TABLE 5 O-mannosylation status of T. reesei strain M403 (pmt1 deletionstrain of MAB01 antibody producing strain, clone 26-8A) from fermenterculture. Percentages calculated from area and intensity of singlecharged signals. In time point d 9 both samples gave 100% to LC, LC +Hex1 being practically absent. 3 d 5 d 6 d 7 d d 8 d 9 d 10 d 11 AverageAverage Std Average Std Average Std Average Average Average Std AverageStd LC 95.8 96.8 0.30 97.5 0.29 97.4 0.36 97.3 100.0 96.6 0.2 95.5 0.11LC + Hex 4.2 3.2 0.30 2.5 0.29 2.6 0.36 2.7 0.0 3.4 0.2 4.5 0.11

TABLE 6 The percentages of area values of three parallel samples fromfermenter cultured M403 from day 7. Area average Std LC 98.5 0.15 LC +Hex 1.5 0.15

TABLE 7 The percentages of intensity values of three parallel samplesfrom fermenter cultured M403 from day 7. Intensity average Std LC 96.30.57 LC + Hex 3.7 0.57

No negative effects of strain growth characteristic and secretioncapacity were observed. The strain M403 grew well and produced increasedamount of antibody in function of time in fermenter culture. The besttiter was obtained from day 10 (Table 8). On day 11 the titer isdecreased.

TABLE 8 Titers from fermenter cultured MAB01 producing strain M403. Theantibody was purified using Protein G 96-well plate. Time point Dayscultured Titer g/l  54:30 hours 2 0.04  71:50 hours 3 0.04  77:45 hours3 0.07 126:20 hours 5 0.91 148:20 hours 6 1.23 168:20 hours 7 1.47192:00 hours 8 1.50 217:15 hours 9 1.35 241:00 hours 10 1.52 275:20hours 11 1.06

Deletion of pmt1 diminished dramatically MAB01 O-mannosylation; theamount of O-mannosylated LC was ˜61% in parental strain, 3% in the bestΔpmt1 clone in shake flask culture and practically 0% in fermenterculture in time point day 9.

Deletion of Pmt1 in a Fab Expressing Trichoderma reesei Strain

The pmt1 disruption cassette (pmt1 amdS) was released from its backbonevector pTTv124 described above by restriction digestion and purifiedthrough gel extraction. Using protoplast transformation the deletioncassette was introduced to T. reesei strains M304 (3-fold proteasedeletion strain expressing MAB01) and M307 (4-fold protease deletionstrain Δpep1 Δtsp1 Δslp1 Δgap1, also described in PCT/EP2013/050126 thathas been transformed to express a Fab). Transformants were plated toacetamidase selective medium (minimal medium containing acetamide as thesole carbon source).

Transformants were screened by PCR for homologous integration of theacetamidase marker to the pmt1 locus using a forward primer outside the5′ flanking region fragment of the construct and the reverse primerinside the AmdS selection marker (5′ integration) as well as a forwardprimer inside the AmdS selection marker and a reverse primer outside the3′ flanking region fragment (3′ integration). Three independenttransformants of each transformation (MAB01 and Fab expressing strains),which gave PCR results displaying correct integration of the constructto the pmt1 locus were selected for single spore purification to obtainuninuclear clones. Proper integration of the disruption cassette wasreconfirmed by PCR using the same primer combinations as described aboveand the absence of the pmt1 gene was verified by using a primercombination targeted to the pmt1 open reading frame. Correct integrationof the disruption cassette was additionally verified for all clonesapplying Southern hybridization. Digested genomic DNA of the threeclones as well as the parental strain were probed against the 5′ and 3′flanks of the pmt1 gene to confirm modification of the pmt1 locus asexpected. Furthermore, the blotted DNA was hybridized with a probespecific to the pmt1 open reading frame in order to substantiate theabsence of pmt1.

MAB01 and Fab Expression for O-Mannosylation Analysis

To evaluate the impact of pmt1 deletion on O-mannosylation levels of mAband Fab molecules, strains were grown in batch fermentations for 7 days,in media containing 2% yeast extract, 4% cellulose, 4% cellobiose, 2%sorbose, 5 g/L KH2PO4, and 5 g/L (NH4)2SO4. Culture pH was controlled atpH 5.5 (adjusted with NH4OH). The starting temperature was 30° C., whichwas shifted to 22° C. after 48 hours. mAb fermentations (strains M304,M403, M406 and M407) were carried out in 4 parallel 2 L glass reactorvessels (DASGIP) with a culture volume of 1 L and the Fab fermentation(TR090 #5) was done in a 15 L steel tank reactor (Infors) with a culturevolume of 6 L. Fab strains (TR090 #5, TR090 #3, TR090 #17) wereadditionally cultured in shake flasks for 4 days at 28° C. Main mediacomponents were 1% yeast extract, 2% cellobiose, 1% sorbose, 15 g/LKH2PO4 and 5 g/L (NH4)2SO4 and the pH was uncontrolled (pH drops from5.5 to <3 during a time course of cultivation). Culture supernatantsamples were taken during the course of the runs and stored at −20° C.Samples were collected daily from the whole course of thesecultivations, and production levels were analyzed by affinity liquidchromatography. Samples with maximum production levels were subject topurification and further O-mannosylation analysis.

Analysis of O-Mannosylation on Fab and mAb

O-mannosylation was analyzed on mAb and Fab molecules expressed fromboth, the pmt1 deletion and parental strains. The mAb and Fab waspurified from culture supernatants using Lambda Select Sure andCaptureSelect Fab Lambda (BAC) affinity chromatography resin,respectively, applying conditions as described by the manufacturesprotocols. Both purified molecules including, the purified mAb and Fabwere subjected to RP-LC-QTOF-MS either as intact and/orreduced/alkylated samples.

For intact analysis, an equivalent of 20 μg protein was injected ontothe column. For reduced/alkylated analyses of mAb, an equivalent of 100μg protein was deglycosylated using PNGase-F enzyme, reduced using DTTand alkylated using iodoacetamide prior to LC-MS analysis. Forreduced/alkylated analyses of Fab, an equivalent of 100 μg protein wasreduced with DTT and alkylated with iodoacetamide prior to LC-MSanalysis. 6 μg of the reduced/alkylated sample were injected onto thecolumn. Reversed-phase chromatography separation was carried out on a2.1×150 mm Zorbax C3 column packed with 5 μm particles, 300 Å pore sizethe eluents were: eluent A 0.1% TFA in water and eluent B 0.1% TFA in70% IPA, 20% ACN, 10% water. The column was heated at 75° C. and the florate was 200 μL/min. The gradient used for the sample separation isshown in Table 9.

TABLE 9 HPLC gradient used for intact and reduced/ alkylated samplesFlow Time % B (mL/min) 0 10 0.1 0.1 10 0.2 2 10 0.2 4 28 0.2 30 36.4 0.231 100 0.2 34 100 0.2 35 10 0.2 40 10 0.2

The HPLC was directly coupled with a Q-TOF Ultima mass spectrometer(Waters, Manchester, UK). The ESI-TOF mass spectrometer was set to runin positive ion mode. The data evaluation of intact andreduced/alkylated analyses was performed using MassLynx analysissoftware (Waters, Manchester, UK). The deconvolution of the averagedmass spectra from the main UV signals was carried out using the MaxEntalgorithm, a part of the MassLynx analysis software (Waters, Manchester,UK). The deconvolution parameters were the following: “max numbers ofiterations” are 8; resolution is 0.1 Da/channel; Uniform Gaussian—widthat half height is 1 Da for intact and 0.5 for the reduced chains andminimum intensity ratios are left 30% and right 30%. The estimated levelof O-mannosylation (%) was determined using the peak signal height afterdeconvolution. The observed O-mannosylation levels (%) of mAbs and Fabsfrom independent pmt1 deletion strains are compared to the ones of therespective parental wild-type strains in Tables 10 and 11.

TABLE10 O-mannosylation level [%] of Fabs from different strains StrainParental Sample M307 TR090#5 TR090#3 TR090#17 Intact Fab 70.1 34.2 34.334.7 LC 58.8 10.4 10.1 10.8 HC 42.9 26.1 25.9 25.8

TABLE 11 O-mannosylation level [%] of MAB01 from different pmt1deficient strains M403, M406 and M407. Parental strain is M304 Strain inyeast extract medium Sample Parental M403 M406 M407 LC 50.7 5.7 5.8 5.8HC 4.8 Not Not Not detected detected detected

The O-mannosylation level was found to be 70% on intact Fab derived fromthe parental strain and reduced to ˜34% in all three pmt1 deletionstrains. The transfer of mannoses was more efficiently diminished on theFab light chains (10% of residual O-mannosylation on light chainsobtained from pmt1 deletion strains vs. 59% for the parental strain), ascompared to the heavy chains, for which it decreased from 43% to ˜26%.

The O-mannosylation level was found to be 50% on the light chain of mAbderived from parental strains and reduced to 5.7-5.8% in all three pmt1deletion strains. The O-mannosylation level was found to be 4.8% on theheavy chain of mAb derived from parental strains and was completelyreduced (below the limit of detection by LC-MS) in all three pmt1deletion strains.

In conclusion, after deletion of pmt1, almost 95% of purified mAb and70% of Fab molecules did no longer contain any O-mannose residues.Therefore, pmt1 is a valuable target to reduce O-mannosylation ofsecreted proteins and to improve product quality of biopharmaceuticalsproduced by Trichoderma reesei.

Example 3: Pmt2 Deletion in a Trichoderma reesei Strain Generation ofPmt2 Deletion Plasmids

Three different deletion plasmids (pTTv34, pTTv122, pTTv186) wereconstructed for deletion of the protein O-mannosyltransferase gene pmt2(TreD22005). All the plasmids contain the same 5′ and 3′ flankingregions for correct integration to the pmt2 locus. The differencebetween the three plasmids is the marker used in the selection; pTTv34contains a gene encoding acetamidase of Aspergillus nidulans (amdS),pTTv122 contains a loopout version (blaster cassette) of the amdS markerand pTTv186 a loopout version (blaster cassette) of a gene encodingorotidine-5′-monophosphate (OMP) decarboxylase of T. reesei (pyr4).

1100 bp of 5′ and 1000 bp of 3′ flanking regions were selected as thebasis of the second protein O-mannosyltransferase gene, pmt2(TreID22005), deletion plasmids. The construction of the first plasmidfor this gene was carried out essentially as described for pmt1 inExample 1. As for pmt1, the first deletion plasmid for pmt2 (plasmidpTTv34, Table 12) used amdS, a gene encoding acetamidase of Aspergillusnidulans, as the selection marker.

Like for pmt1 in Example 1, to clone the second deletion plasmid,pTTv122 (Table 12), the amdS marker was removed from the deletionplasmid pTTv34 with NotI digestion and replaced by amdS blaster cassettefor which the fragments were produced by PCR (see Example 1 above fordetails). The plasmid pTTv122 was constructed using the yeastrecombination system described in Example 1. The plasmid DNA from theyeast transformants was rescued by transformation into Escherichia coli.A few clones were cultivated, plasmid DNA was isolated and digested toscreen for correct recombination using standard laboratory methods. Afew clones with correct insert sizes were sequenced and stored.

The third deletion plasmid for pmt2, pTTv186 (Table 12) was cloned likethe third plasmid for pmt1; the amdS blaster cassette was removed fromthe deletion plasmid pTTv122 with NotI digestion and replaced by thepyr4 blaster cassette described in Example 1. The pyr4 blaster cassettewas obtained from another plasmid with NotI digestion, ligated to NotIcut pTTv122 and transformed into E. coli using standard laboratorymethods. A few transformants were cultivated, plasmid DNA isolated anddigested to screen for correct ligation and orientation of the pyr4blaster cassette using standard laboratory methods. One clone withcorrect insert size and orientation was sequenced and stored. Thesedeletion plasmids for pmt2 (pTTv34, pTTv122 and pTTv186, Table 12)result in 3186 bp deletion in the pmt2locus and cover the completecoding sequence of PMT2.

TABLE 12 Primers for generating deletion plasmidspTTv34, pTTv122 and pTTv186 for proteinO-mannosyltransferase 2 (pmt2, TreID22005). Primer SequenceDeletion plasmid pTTv34 for pmt2 (TreID22005), vector backbone pRS42622005_5′F CGATTAAGTTGGGTAACGCCAGGGTT TTCCCAGTCACGACGGTTTAAACGTTTCAGGTACCAACACCTG (SEQ ID NO: 70) 22005_5′R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGTGCGGCCGCGGC GAAGAGTCTGGCGGGGA (SEQ ID NO: 71) 22005_3′FCGGTTCTCATCTGGGCTTGCTCGGTC CTGGCGTAGATCTAGCGGCCGCAAGA GGATGGGGGTAAAGCT(SEQ ID NO: 72) 22005_3′R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGTTTAAACGAG GAGGACTCGTGAGTTAT (SEQ ID NO: 73)Deletion plasmid pTTv122 for pmt2 (TreID22005), vector backbone pTTv34T280_22005_amds_5for GCGCCCTTCCGCCTCGACAATCCCCGCCAGACTCTTCGCCGCGGCCGCGGCC GGCCGCGATCGCCTAGATCTACGCCAGGACCG (SEQ ID NO: 74) T283_amds_3rev_loop CGGTCCTGGCGTAGATCTAGGGCGCGCCACTGGAAACGCAACCCTGAA (SEQ ID NO: 75) T284_amds_loop_5forTTCAGGGTTGCGTTTCCAGTGGCGCG CCCTAGATCTACGCCAGGACCG (SEQ ID NO: 76)T285_22005_loop_3rev GAGCTGGCCAGAAAAGACCAAGCTTTACCCCCATCCTCTTGCGGCCGCGATT ATTGCACAAGCAGCGA (SEQ ID NO: 77)Deletion plasmid pTTv186 for pmt2 (TreID22005), vector backbone pTTv122no new primers, pTTv122 digested with NotI andligated with pyr4-loopout fragment from another plasmid

Generation of Pmt2 Deletion Strains M338, M339 and M340

To remove vector sequence plasmid pTTv122 (Δpmt2-amdS) was digested withPmeI+XbaI and the 5.2 kb fragment purified from agarose gel usingQAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the pmt2deletion cassette was used to transform the strain M124 (M124 strain isdescribed in WO2012/069593). Protoplast preparation and transformationwere carried out essentially according to Penttilä et al., 1987, Gene61:155-164 and Gruber et al, 1990, Current Genetics 18:71-76 for amdSselection.

120 colonies were picked as selective streaks. 10 transformants werescreened by PCR using the primers in Table 13 for the correctintegration of the deletion cassette using standard laboratory methods.Five putative deletion clones were purified to single cell clones.Purified clones (two parallel from each) were rescreened for correctintegration and for deletion of pmt2 ORF (primers on Table 13). Fiveclones were selected for Southern analyses.

TABLE 13 Primers for screening integrationof deletion cassette pTTv122 and fordeletion of protein O-mannosyltransferase 2(pmt2, TreID22005) from M124. Primer Sequence T288_22005_5intACGAGTTGTTTCGTGTACCG (SEQ ID NO: 78) T020_Amds_rev2CTTTCCATTCATCAGGGATGG (SEQ ID NO: 79) T021_amds_end_fwdGGAGACTCAGTGAAGAGAGG (SEQ ID NO: 80) T289_22005_3int ATGTTGCAGTTGCGAAAG(SEQ ID NO: 81) T290_22005_5orf CCCTCGTCGCAGAAAAGATG (SEQ ID NO: 82)T291_22005_3orf AGCCTCCTTGGGAACCTCAG (SEQ ID NO: 83)

Deletion of pmt2 was verified by Southern analyses. DNA for Southernanalyses was extracted with Easy-DNA kit for genomic DNA isolation(Invitrogen) essentially according to the manufacturer's instructions.

Southern analyses were essentially performed as described in Example 1.Fragments for probes were produced by PCR using the primers listed inTable 14 using a T. reesei strain M124 as the template for the ORF probeand plasmid pTTv122 for the 5′ and 3′ flank probes. PCR products wereseparated with agarose gel electrophoresis and correct fragments wereisolated from the gel with a gel extraction kit (Qiagen) using standardlaboratory methods.

TABLE 14 Primers for production of probe fragmentsused in Southern analyses of proteinO-mannosyltransferase 2 (pmt2, TreID22005) deletion strains. PrimerSequence T639_22005 5′ flank CTTAGTGCGGCTGGAGGGCG probe F(SEQ ID NO: 84) T640_22005 5′ flank GGCCGGTTCGTGCAACTGGA probe R(SEQ ID NO: 85) T641_22005 3′ flank GGCCGCAAGAGGATGGGGGT probe F(SEQ ID NO: 86) T642_22005 3′ flank TCGGGCCAGCTGAAGCACAAC probe R(SEQ ID NO: 87) T643_22005 orf 5′ probe TTGAGGAACGGCTGCCTGCG(SEQ ID NO: 88) T644_22005 orf 3′ probe CGATGGCTCCGTCATCCGCC(SEQ ID NO: 89)

Three of the clones did not hybridise with pmt2 ORF probe (Data notshown) indicating successful deletion of pmt2. Analyses using 5′ and 3′flank probes revealed that the same three clones were single integrants(Data not shown). The two other clones (19-35A and 19-40B) gave signalscorresponding to parental strain M124. Three pure clones have beenstored for future use (M338; 19-7B, M339; 19-22B and M340; 19-39B).

Analyses of Δpmt2 strains M338, M339 and M340 Shake flask cultivation ofT. reesei strain M124 and the pmt2 deletion strains (19-7B/M338,19-22B/M339 and 19-39B/M340) was carried out in Trichoderma minimalmedium with 40 g/l lactose, 20 g/l spent grain extract, 100 mM PIPPS, pH5.5 with and without 1 M sorbitol as osmotic stabiliser at +28° C., 200rpm. Samples were collected on days 3, 5 and 7 by vacuum filtration.Supernatant samples were stored to −20° C. (antibody and glycananalyses) or used in pH determinations. Mycelia for cell dry weightdeterminations were rinsed once with DDIW and dried at +100° C. for20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIWand stored to −20° C.

Generation of Pmt2 Deletion Strains M452, M453 and M454

Generation of M317 is described in Example 1 above.

To remove vector sequence plasmid pTTv186 (Δpmt2-pyr4) was digested withPmeI+XbaI and the 4.1 kb fragment purified from agarose gel usingQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the pmt2deletion cassette was used to transform M317.

Protoplast preparation and transformation were carried out essentiallyaccording to Penttilä et al., 1987, Gene 61:155-164 and Gruber et al,1990, Current Genetics 18:71-76 for pyr4 selection.

100 colonies were picked as selective streaks. 20 transformants werescreened by PCR using the primers in Table 15 for the correctintegration of the deletion cassette using standard laboratory methods.Nine putative deletion clones were purified to single cell clones.Purified clones were rescreened for 5′ integration and for deletion ofpmt2 ORF (primers on Table 14). Three clones were pure deletants (i.e.no signal with ORF primers).

TABLE 15 Primers for screening integration ofdeletion cassette pTTv186 and for deletionof protein O-mannosyltransferase 2 (pmt2, TreID22005) from M317. PrimerSequence T288_22005_5int ACGAGTTGTTTCGTGTACCG (SEQ ID NO: 90)T027_Pyr4_orf_start_rev TGCGTCGCCGTCTCGCTCCT (SEQ ID NO: 91)T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 92)T289_22005_3int ATGTTGCAGTTGCGAAAG (SEQ ID NO: 93) T290_22005_5orfCCCTCGTCGCAGAAAAGATG (SEQ ID NO: 94) T291_22005_3orfAGCCTCCTTGGGAACCTCAG (SEQ ID NO: 95)

Deletion of pmt2 was verified by Southern analyses. DNA for Southernanalyses was extracted with Easy-DNA kit for genomic DNA isolation(Invitrogen) essentially according to the manufacturer's instructions.

Southern analyses were essentially performed as described in Example 1.Fragments for probes were produced by PCR using the primers listed inTable 16 using a T. reesei wild type strain QM6a (ATCC13631) as thetemplate for pmt2 ORF probe and plasmid pTTv186 for 5′ and 3′ flankprobes. PCR products were separated with agarose gel electrophoresis andcorrect fragments were isolated from the gel with a gel extraction kit(Qiagen) using standard laboratory methods.

TABLE 16 Primers for production of probe fragmentsused in Southern analyses of proteinO-mannosyltransferase 2 (pmt2, TreID22005) deletion clones. PrimerSequence T639_22005 5′ flank CTTAGTGCGGCTGGAGGGCG probe F(SEQ ID NO: 96) T640_22005 5′ flank GGCCGGTTCGTGCAACTGGA probe R(SEQ ID NO: 97) T641_22005 3′ flank GGCCGCAAGAGGATGGGGGT probe F(SEQ ID NO: 98) T642_22005 3′ flank TCGGGCCAGCTGAAGCACAAC probe R(SEQ ID NO: 99) T290_22005_5orf CCCTCGTCGCAGAAAAGATG (SEQ ID NO: 100)T291_22005_3orf AGCCTCCTTGGGAACCTCAG (SEQ ID NO: 101)

None of the clones hybridised with pmt2 ORF probe (Data not shown)indicating successful deletion of pmt2. Analyses using 5′ and 3′ flankprobes revealed that two of the clones were single integrants (Data notshown). One clone gave additional signal from the 3′flank probing (Datanot shown) and thus indicated partial or multiple integration of thedeletion cassette. Three pure clones (with and without additional copiesof the deletion cassette) have been stored for future use (M452; 27-10A,M453; 27-17A and M454: 27-18B).

Analyses of ΔPmt2 Strains M452, M453 and M454

Shake flask cultivation of T. reesei strain M304 and three pmt2 deletionstrains (27-10A/M452, 27-17A/M453 and 27-18B/M454) was carried out inTrichoderma minimal medium with 40 g/l lactose, 20 g/l spent grainextract, 100 mM PIPPS, 9 g/l casamino acids, pH 5.5 at +28° C., 200 rpm.Samples were collected on days 3, 5, 7 and 10 by vacuum filtration.Supernatant samples were stored to −20° C. (antibody and glycananalyses) or used in pH determinations. Mycelia for cell dry weightdeterminations were rinsed once with DDIW and dried at +100° C. for20-24 h. Mycelia for genomic DNA extraction were rinsed once with DDIWand stored to −20° C.

O-mannosylation level analysis was performed to pmt2 deletion strains asto flask cultures of pmt1 deletion strains. No difference was observedin O-mannosylation compared to parental strain M304.

Example 4: Pmt3 Deletion in a Trichoderma reesei Strain Generation ofPmt3 Deletion Plasmids

Three different deletion plasmids (pTTv35, pTTv123, pTTv187) wereconstructed for deletion of the protein O-mannosyltransferase gene pmt3(TreD22527). All the plasmids contain the same 5′ and 3′ flankingregions for correct integration to the pmt3 locus. The differencebetween the three plasmids is the marker used in the selection; pTTv35contains a gene encoding acetamidase of Aspergillus nidulans (amdS),pTTv123 contains a loopout version (blaster cassette) of the amdS markerand pTTv187 a loopout version (blaster cassette) of a gene encodingorotidine-5′-monophosphate (OMP) decarboxylase of T. reesei (pyr4).

1100 bp of 5′ and 1000 bp of 3′ flanking regions were selected as thebasis of the third protein O-mannosyltransferase gene, pmt3(TreID22527), deletion plasmids. The construction of the first plasmidfor this gene was carried out essentially as described for pmt1 inExample 1. As for pmt1, the first deletion plasmid for pmt3 (plasmidpTTv35, Table 17) used amdS, a gene encoding acetamidase of Aspergillusnidulans, as the selection marker.

Like for pmt1 in Example 1, to clone the second deletion plasmid,pTTv123 (Table 16), the amdS marker was removed from the deletionplasmid pTTv35 with NotI digestion and replaced by amdS blaster cassettefor which the fragments were produced by PCR (see Example 1 above fordetails). The plasmid pTTv123 was constructed using the yeastrecombination system described in Example 1. The plasmid DNA from theyeast transformants was rescued by transformation into Escherichia coli.A few clones were cultivated, plasmid DNA was isolated and digested toscreen for correct recombination using standard laboratory methods. Afew clones with correct insert sizes were sequenced and stored.

The third deletion plasmid for pmt3, pTTv187 (Table 17) was cloned likethe third plasmid for pmt1; the amdS blaster cassette was removed fromthe deletion plasmid pTTv123 with NotI digestion and replaced by thepyr4 blaster cassette described in Example 1. The pyr4 blaster cassettewas obtained from another plasmid with NotI digestion, ligated to NotIcut pTTv123 and transformed into E. coli using standard laboratorymethods. A few transformants were cultivated, plasmid DNA isolated anddigested to screen for correct ligation and orientation of the pyr4blaster cassette using standard laboratory methods. One clone withcorrect insert size and orientation was sequenced and stored. Thesedeletion plasmids for pmt3 (pTTv35, pTTv123 and pTTv187, Table 17)result in 2495 bp deletion in the pmt3locus and cover the completecoding sequence of PMT3.

TABLE 17 Primers for generating deletion plasmidspTTv35, pTTv123 and pTTv187 for proteinO-mannosyltransferase 3 (pmt3, TreID22527). Primer SequenceDeletion plasmid pTTv35 for pmt3 (TreID22527), vector backbone pRS42622527_5′F CGATTAAGTTGGGTAACGCCAGGGTTT TCCCAGTCACGACGGTTTAAACGTGTTTAAATTTGATGAGGC (SEQ ID NO: 102) 22527_5′R ATCTCTCAAAGGAAGAATCCCTTCAGGGTTGCGTTTCCAGTGCGGCCGCGGTCT CAGAGACAGCCTTCT (SEQ ID NO: 103) 22527_3′FCGGTTCTCATCTGGGCTTGCTCGGTCC TGGCGTAGATCTAGCGGCCGCACTCGG CTTCTTTGTCCGAG(SEQ ID NO: 104) 22527_3′R GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCGTTTAAACTCCTC GTCGGCAACAAGGCC (SEQ ID NO: 105)Deletion plasmid pTTv123 for pmt3 (TreID22527), vector backbone pTTv35T281_22527_amds_5for GCAGATCTGGGGGAGGAATCAGAAGGCTGTCTCTGAGACCGCGGCCGCGGCCGG CCGCGATCGCCTAGATCTACGCCAGGACCG (SEQ ID NO: 106) T283_amds_3rev_loop CGGTCCTGGCGTAGATCTAGGGCGCGCCACTGGAAACGCAACCCTGAA (SEQ ID NO: 107) T284_amds_loop_5forTTCAGGGTTGCGTTTCCAGTGGCGCGC CCTAGATCTACGCCAGGACCG (SEQ ID NO: 108)T286_22527_loop_3rev AAAGTGGGCGAGCTGAGATACTCGGACAAAGAAGCCGAGTGCGGCCGCGATTAT TGCACAAGCAGCGA (SEQ ID NO: 109)Deletion plasmid pTTv187 for pmt3 (TreID22527), vector backbone pTTv123no new primers, pTTv123 digested with NotIand ligated with pyr4-loopout fragment from another plasmid.

Generation of Pmt3 Deletion Strains M341 and M342

To remove vector sequence plasmid pTTv123 (Δpmt3-amdS) was digested withPmeI+XbaI and the 5.2 kb fragment purified from agarose gel usingQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the pmt3deletion cassette was used to transform the strain M124. Protoplastpreparation and transformation were carried out essentially according toPenttilä et al., 1987, Gene 61:155-164 and Gruber et al, 1990, CurrentGenetics 18:71-76 for amdS selection.

120 colonies were picked as selective streaks. 10 transformants werescreened by PCR using the primers in Table 18 for the correctintegration of the deletion cassette using standard laboratory methods.Three putative deletion clones were purified to single cell clones.Purified clones (three parallel from each) were rescreened for correctintegration and for deletion of pmt3 ORF (primers on Table 18). Threeclones were selected for Southern analyses.

TABLE 18 Primers for screening integration ofdeletion cassette pTTv123 and for deletion ofprotein O-mannosyltransferase 3 (pmt3, TreID22527) from M124. PrimerSequence T292_22527_5int ACGGGAGATCTCGGAAAA (SEQ ID NO: 110)T020_Amds_rev2 CTTTCCATTCATCAGGGATGG (SEQ ID NO: 111) T021_amds_end_fwdGGAGACTCAGTGAAGAGAGG (SEQ ID NO: 112) T293_22527_3int ATGAAGCTCAGCCTGTGG(SEQ ID NO: 113) T294_22527_5orf GGGGACGGCTTGAGGAAG (SEQ ID NO: 114)T295_22527_3orf CTGCTTGCTGCTTCCAGTCA (SEQ ID NO: 115)

Deletion of pmt3 was verified by Southern analyses. DNA for Southernanalyses was extracted with Easy-DNA kit for genomic DNA isolation(Invitrogen) essentially according to the manufacturer's instructions.

Southern analyses were essentially performed as described in Example 1.Fragments for probes were produced by PCR using the primers listed inTable 19 using a T. reesei strain M124 as the template for the ORF probeand plasmid pTTv123 for the 5′ and 3′ flank probes. PCR products wereseparated with agarose gel electrophoresis and correct fragments wereisolated from the gel with a gel extraction kit (Qiagen) using standardlaboratory methods.

TABLE 19 Primers for production of probe fragmentsused in Southern analyses of proteinO-mannosyltransferase 3 (pmt3, TreID22527) deletion strains. PrimerSequence T645_22527 5′ flank TGGCAGATGCCGAAAGGCGG probe F(SEQ ID NO: 116) T646_22527 5′ flank TGGCAACCAGCTGTGGCTCC probe R(SEQ ID NO: 117) T647_22527 3′ flank CGGCCGCACTCGGCTTCTTT probe F(SEQ ID NO: 118) T648_22527 3′ flank GAGTGGGCTAGGCGCAACGG probe R(SEQ ID NO: 119) T649_22527 orf 5′ probe GGATCGGCCACTGCCACCAC(SEQ ID NO: 120) T650_22527 orf 3′ probe GCCCACTTCTCTGCGCGTGT(SEQ ID NO: 121)

Two of the clones did not hybridise with pmt3 ORF probe (Data not shown)indicating successful deletion of pmt3. Analyses using 5′ and 3′ flankprobes revealed that the same two clones were single integrants (Datanot shown). One clone (20-32C) gave signals corresponding to parentalstrain M124. Two clones have been stored for future use (M341; 20-34Cand M342; 20-35B).

Analyses of ΔPmt3 Strains M341 and M342

Shake flask cultivation of T. reesei strain M124 and the pmt3 deletionstrains (20-34C/M341 and 20-35B/M342) was carried out in Trichodermaminimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mMPIPPS, pH 5.5 with and without 1 M sorbitol as osmotic stabiliser at+28° C., 200 rpm. Samples were collected on days 3, 5 and 7 by vacuumfiltration. Supernatant samples were stored to −20° C. (antibody andglycan analyses) or used in pH determinations. Mycelia for cell dryweight determinations were rinsed once with DDIW and dried at +100° C.for 20-24 h. Mycelia for genomic DNA extraction were rinsed once withDDIW and stored to −20° C.

Generation of Pmt3 Deletion Strains M522 and M523

Generation of M317 is described in Example 1 above.

To remove vector sequence plasmid pTTv187 (Δpmt3-pyr4) was digested withPmeI+XbaI and the 4.1 kb fragment purified from agarose gel usingQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the pmt3deletion cassette was used to transform M317. Protoplast preparation andtransformation were carried out essentially according to Penttilä etal., 1987, Gene 61:155-164 and Gruber et al, 1990, Current Genetics18:71-76 for pyr4 selection.

200 colonies were picked as selective streaks. 59 transformants werescreened by PCR using the primers in Table 20 for the correctintegration of the deletion cassette using standard laboratory methods.Three putative deletion clones were purified to single cell clones.Purified clones were rescreened for correct integration and for deletionof pmt3 ORF (primers on Table 19). Two clones (several parallels) werepure deletants (i.e. no signal with ORF primers).

TABLE 20 Primers for screening integration ofdeletion cassette pTTv187 and fordeletion of protein O-mannosyltransferase 3(pmt3, TreID22527) from M317. Primer Sequence T292_22527_5intACGGGAGATCTCGGAAAA (SEQ ID NO: 122) T026_Pyr4_orf_5rev2CCATGAGCTTGAACAGGTAA (SEQ ID NO: 123) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 124) T293_22527_3int ATGAAGCTCAGCCTGTGG(SEQ ID NO: 125) T649_22527 orf 5′ GGATCGGCCACTGCCACCAC probe(SEQ ID NO: 126) T650_22527 orf 3′ GCCCACTTCTCTGCGCGTGT probe(SEQ ID NO: 127)

Deletion of pmt3 was verified by Southern analyses. DNA for Southernanalyses was extracted with Easy-DNA kit for genomic DNA isolation(Invitrogen) essentially according to the manufacturer's instructions.

Southern analyses were essentially performed as described in Example 1.Fragments for probes were produced by PCR using the primers listed inTable 21 using a T. reesei wild type strain QM6a (ATCC13631) as thetemplate for the ORF probe and plasmid pTTv187 for the 5′ and 3′ flankprobes. PCR products were separated with agarose gel electrophoresis andcorrect fragments were isolated from the gel with a gel extraction kit(Qiagen) using standard laboratory methods.

TABLE 21 Primers for production of probefragments used in Southern analyses of protein O-mannosyltransferase 3(pmt3, TreID22527) deletion strains. Primer Sequence T645_22527 5′ flankTGGCAGATGCCGAAAGGCGG probe F (SEQ ID NO: 128) T646_22527 5′ flankTGGCAACCAGCTGTGGCTCC probe R (SEQ ID NO: 129) T647_22527 3′ flankCGGCCGCACTCGGCTTCTTT probe F (SEQ ID NO: 130) T648_22527 3′ flankGAGTGGGCTAGGCGCAACGG probe R (SEQ ID NO: 131) T874_pmt3_orf_f3CTCTGCGCGTGTTGTGG (SEQ ID NO: 132) T875_pmt3_orf_r3 TAAGGGTGCGGATTCGG(SEQ ID NO: 133)

Eight of the clones did not hybridise with pmt3 ORF probe (Data notshown) indicating successful deletion of pmt3. One clone (33-37K)hybridised with pmt3 ORF probe even though the signal size did notcorrespond to those from parental strains suggesting rearrangement inthe pmt3locus. Analyses using 5′ and 3′ flank probes revealed that theeight Δpmt3 clones were single integrants (Data not shown). One clone(33-37K) gave incorrect or additional signals suggesting rearrangementsin the pmt3 locus and multiple integrations of the deletion cassette.Two pure clones have been stored for future use (M522; 33-34A and M523;33-188A-a).

Analyses of Δpmt3 strains M522 and M523

24-well plate cultivation of T. reesei strain M304 and eight pmt3deletion strains (33-34S/M522, 33-34T, 33-34U, 33-340, 33-188A-a/M523,33-188B-a, 33-188C-a and 33-188D-a) was carried out in Trichodermaminimal medium with 40 g/l lactose, 20 g/l spent grain extract, 100 mMPIPPS, 9 g/l casamino acids, pH 5.5 at +28° C., 800 rpm with humiditycontrol. Samples were collected on days 3, 5 and 6 by centrifugation.Supernatant samples were stored to −20° C. Mycelia for cell dry weightdeterminations were rinsed once with DDIW and dried at +100° C. for20-24 h. Mycelia for genomic DNA extraction were rinsed twice with DDIWand stored to −20° C.

O-mannosylation level analysis was performed to pmt3 deletion strains asto flask cultures of pmt1 deletion strains. No difference was observedin O-mannosylation compared to parental strain M304.

Example 5—Pmt Homologs

T. reesei pmt Homologs were Identified from Other Organisms.

BLAST searches were conducted using the National Center forBiotechnology Information (NCBI) non-redundant amino acid database usingthe Trichoderma reesei PMT amino acid sequences as queries. Sequencehits from the BLAST searches were aligned using the ClustalW2 alignmenttool provided by EBI. Phylogenetic trees were generated using averagedistance with BLOSUM62 after aligning the sequences in the Clustal Omegaalignment tool.

A phylogenetic tree and a partial sequence alignment of the results ofthe PMT BLAST searches are depicted in FIGS. 5 and 6, respectively.

1-16. (canceled)
 17. A Protein O-mannosyltransferase (PMT)-deficient filamentous fungal cell comprising a) a first mutation in a gene encoding an endogenous protease that eliminates an endogenous protease activity as compared to a parental filamentous fungal cell which does not have said first mutation; b) a second mutation in a PMT gene that reduces endogenous O-mannosyltransferase activity compared to a parental filamentous fungal cell which does not have said second mutation; and c) a polynucleotide encoding a light chain of an antibody and a polynucleotide encoding a heavy chain of an antibody; wherein: the antibody is secreted by the PMT-deficient filamentous fungal cell upon expression of the polynucleotides encoding the light chain and the heavy chain of the antibody; said antibody comprising reduced O-mannosylation on the light chain, said reduced O-mannosylation being less than about 10% that of a light chain secreted by said parental filamentous fungal cell which does not have said second mutation, O-mannosylation being defined as mole % of mannose residues per polypeptide chain; and said filamentous fungal cell is a Trichoderma or Myceliophthora cell.
 18. The PMT-deficient filamentous fungal cell of claim 17, wherein said second mutation that reduces the endogenous O-mannosyltransferase activity is a deletion or a disruption of a PMT gene encoding the endogenous protein O-mannosyltransferase activity.
 19. The PMT-deficient filamentous fungal cell of claim 17, wherein said second mutation in a PMT gene is a mutation in either: a) PMT1 gene comprising the polynucleotide of SEQ ID NO:1, b) a functional homologous gene of PMT1 gene, which functional homologous gene is capable of restoring parental O-mannosylation level by functional complementation when introduced into a T. reesei strain having a disruption in said PMT1 gene, or c) a polynucleotide encoding a polypeptide having at least 50% identity with SEQ ID NO:2, said polypeptide having O-mannosyltransferase activity.
 20. The PMT-deficient filamentous fungal cell of claim 17, wherein the reduced endogenous O-mannosyltransferase activity results from a deletion or a disruption of a PMT1 gene encoding a polypeptide of SEQ ID NO:2 or SEQ ID NO:12.
 21. The PMT-deficient filamentous fungal cell of claim 17, wherein said cell has a third mutation that reduces or eliminates the level of expression of a dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (ALG3) gene compared to the level of expression in a parental cell which does not have such third mutation.
 22. The PMT-deficient filamentous fungal cell of claim 17, further comprising one or more polynucleotides encoding a polypeptide selected from the group consisting of: a) α1,2 mannosidase; b) N-acetylglucosaminyltransferase I catalytic domain; c) α mannosidase II; d) N-acetylglucosaminyltransferase II catalytic domain; e) β1,4 galactosyltransferase; and, f) fucosyltransferase.
 23. The PMT-deficient filamentous fungal cell of claim 17, wherein said cell is a Trichoderma cell comprising a mutation that reduces or eliminates the protein O-mannosyltransferase activity of Trichoderma pmt1.
 24. The PMT-deficient filamentous fungal cell of claim 17, wherein said cell is a Trichoderma reesei cell, and said cell comprises mutations that reduce or eliminate the activity of a) the three endogenous proteases pep1, tsp1 and slp1; b) the three endogenous proteases gap1, slp1 and pep1; c) the three endogenous proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1 and gap2; d) three to six proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1 and gap2; or e) seven to ten proteases selected from the group consisting of pep1, pep2, pep3, pep4, pep5, pep7, pep8, tsp1, slp1, slp2, slp3, slp5, slp6, slp7, slp8, tpp1, gap1 and gap2.
 25. The PMT-deficient filamentous fungal cell of claim 17, wherein the O-mannosylation level on the expressed light chain of the antibody is reduced to 0%.
 26. The PMT-deficient filamentous fungal cell of claim 17, wherein said produced antibody is a mammalian antibody selected from the group consisting of: a) an immunoglobulin, such as IgG, b) a light chain or heavy chain of an immunoglobulin, c) a heavy chain or a light chain of an antibody, d) a single chain antibody, e) a camelid antibody, f) a monomeric or multimeric single domain antibody, g) a Fab-fragment, a Fab₂-fragment, and, h) their antigen-binding fragments.
 27. A method for producing an antibody having reduced O-mannosylation, comprising: a) providing the PMT-deficient filamentous fungal cell of claim 17, and b) culturing the cell to produce said antibody, consisting of heavy and light chains, having reduced O-mannosylation.
 28. The method of claim 27, wherein said produced antibody is a mammalian antibody selected from the group consisting of: a) an immunoglobulin, optionally IgG; b) a light chain or heavy chain of an immunoglobulin; c) a heavy chain or a light chain of an antibody; d) a single chain antibody; e) a camelid antibody; f) a monomeric or multimeric single domain antibody; g) a Fab-fragment, a Fab₂-fragment; and, h) an antigen-binding fragment of one of a) through g). 